Research Article Assessment of Functional EST-SSR Markers...

Research ArticleAssessment of Functional EST-SSR Markers(Sugarcane) in Cross-Species Transferability Genetic Diversityamong Poaceae Plants and Bulk Segregation Analysis

Shamshad Ul Haq123 Pradeep Kumar14 R K Singh1 Kumar Sambhav Verma5

Ritika Bhatt23 Meenakshi Sharma3 Sumita Kachhwaha3 and S L Kothari235

1Biotechnology Division UP Council of Sugarcane Research Shahjahanpur 242001 India2Interdisciplinary Programme of Life Science for Advance Research and Education University of Rajasthan Jaipur 302004 India3Department of Botany University of Rajasthan Jaipur 302015 India4School of Biotechnology Yeungnam University Gyeongsan 712-749 Republic of Korea5Amity Institute of Biotechnology Amity University Rajasthan Jaipur 302006 India

Correspondence should be addressed to Shamshad Ul Haq shamshadbiotechgmailcom

Received 26 November 2015 Revised 13 April 2016 Accepted 26 April 2016

Academic Editor Norman A Doggett

Copyright copy 2016 Shamshad Ul Haq et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Expressed sequence tags (ESTs) are important resource for gene discovery gene expression and its regulation molecular markerdevelopment and comparative genomics We procured 10000 ESTs and analyzed 267 EST-SSRs markers through computationalapproach The average density was one SSR1045 kb or 64 frequency wherein trinucleotide repeats (6674) were the mostabundant followed by di- (2610) tetra- (467) penta- (15) and hexanucleotide (12) repeats Functional annotations weredone and after-effect newly developed 63 EST-SSRs were used for cross transferability genetic diversity and bulk segregationanalysis (BSA) Out of 63 EST-SSRs 42 markers were identified owing to their expansion genetics across 20 different plants whichamplified 519 alleles at 180 loci with an average of 288 alleleslocus and the polymorphic information content (PIC) ranged from051 to 093 with an average of 083The cross transferability ranged from 25 for wheat to 9722 for Schlerostachya with an averageof 5586 and genetic relationships were established based on diversification among themMoreover 10 EST-SSRs were recognizedas important markers between bulks of pooled DNA of sugarcane cultivars through BSA This study highlights the employabilityof the markers in transferability genetic diversity in grass species and distinguished sugarcane bulks

1 Introduction

Sugarcane is a bioenergy crop belonging to the genus Sac-charum L of the tribe Andropogoneae (family Poaceae)This tribe comprises grass species which have high eco-nomic value The noble sugarcane varieties are developedfrom interspecific hybridization of Saccharum officinarum L(2119899 = 80) which has high sugar content with less diseasetolerance and Saccharum spontaneum (2119899 = 40 to 120) whichprovides stress disease tolerance and high fiber content forbiomassThe taxonomy and genetic constitution of sugarcaneare complicated due to complex interspecific aneupolyploidgenome which makes chromosome numbers range from 100

to 130 [1] Moreover six Saccharum spp (S spontaneum Sofficinarum S robustum S edule S barberi and S sinense)and four Saccharum related genera (Erianthus MiscanthusSclerostachya and Narenga) have purportedly undergoneinterbreeding forming the ldquoSaccharum complexrdquo [2 3] Theinterbreeding has made their genome more complex andadded tomultigenic andor multiallelic nature for most agro-nomic traits that made sugarcane breeding a more difficulttask [4]

A vast array of genomic tools has been developed whichhas opened new ways to define the genetic architecture ofsugarcane and helped to explore its functional system [15] Among the molecular markers microsatellites are most

Hindawi Publishing CorporationGenetics Research InternationalVolume 2016 Article ID 7052323 16 pageshttpdxdoiorg10115520167052323

2 Genetics Research International

favored for a variety of genetic applications due to theirmultiallelic nature high reproducibility cross transferabilitycodominant inheritance abundance and extensive genomecoverage [6ndash8] Microsatellites or simple sequences repeats(SSRs) are monotonous repetitions of very short (one tosix) nucleotide motifs which occur as interspersed repet-itive elements in all eukaryotic and prokaryotic genomesHowever transcribed regions of the genome also containenormous range of microsatellites that correspond to genicmicrosatellites or EST-SSRs Therefore expressed sequencetags (ESTs) are the short transcribed portions and involvedin the variety of metabolic functions The presence of themicrosatellites in genes as well as ESTs unveils the biologicalsignificance of SSR distribution expansion and contractionon the function of the genes themselves [9]

Presently huge amounts of expressed sequence tagshave been deposited in public database (NCBI) In silicoapproaches to retrieve EST sequences from NCBI and func-tional annotations provide more constructive EST-SSRs orgene-based SSR (genic SSRs) marker development besidesown EST libraries developmentThis method of the EST-SSRmarkers development provides the easiest way to reduce costtime and labours along with more meaningful marker iden-tifications [10] The presence of microsatellites in the genicregion is found to be more conserved due to which they pos-sess high reproducibility and high interspecificintraspecifictransferability Hence EST-SSR could be used for polymor-phism genetic diversity cross transferability and compara-tive mapping in different plant species Accordingly severalgenetic studies were done on sugarcane using microsatellitemarkers to decipher polymorphism cross transferabilitygenetic diversity informative marker detection through bulksegregation analysis (BSA) and comparative genomics [811ndash13] The objective of the present study was to retrieveEST sequences for more informative EST-SSR developmentand their genetic assessment within and across the taxathrough cross transferability genetic relationships and bulksegregation analysis

2 Materials and Methods

21 EST Sequences Retrieving ESTs Assembling andMicrosatellites Identification Total 10000 EST sequences ofthe Saccharum spp were downloaded in Fasta format fromNational Centre for Biotechnology Information (NCBI) formicrosatellites deciphering Further ESTs assembling wascarried out using CAP3 programme (httpmobylepasteurfrcgi-binportalpyformscap3) for minimization of se-quences redundancyMicrosatellite identificationwas carriedout using MISA software (httppgrcipk-gaterslebendemisa) and the criteria for SSR detection were 6 4 3 3and 3 repeat units for di- tri- tetra- penta- and hexanu-cleotides respectively SSR primer pairs (forward andreverse) were designed for the selected EST sequences havingmicrosatellites using online web tool batch primer 3 pipeline[14]

22 EST-SSR Sequences Annotation Assessment of ESTsequences having SSR was done through blastnblastx

analysis for homology search and against nonredundant (nr)protein at the NCBI Furthermore functional annotationpipeline was also run at online tool for gene ontology (GO)which was intended for different GO functional classeslike biological process cellular component and molecularfunction [15]

23 PCR Amplification and Electrophoresis PCR reactionswere carried out in a total of 10 120583L volume containing 25 ngtemplate DNA 10 120583L (10 pmol120583L) of each forward andreverse primer 100mM of dNTPs 05U of Taq DNA poly-merase and 10 120583L of 10x PCR buffer with 25mM of MgCl

2

Amplification was performed in a thermal cycler (Bio-Rad)in the following conditions initial denaturation at 94∘C for5min followed by 30 amplification cycles of denaturation for1min at 94∘C followed by annealing temperature (119879

119886) for

1min and then extension for 2min at 72∘C final extension at72∘C for 7min was allowed The PCR conditions particularlythe annealing temperatures (varying from 52∘C to 58∘C) foreach primer were standardized and amplified products werestored at 4∘C The PCR products were analyzed on a 7native PAGE in vertical gel electrophoresis unit (BangaloreGenei) using TBE buffer The sizes of amplified fragmentswere estimated using 50 bp DNA ladder (Fermentas) Gelswere documented using ethidium bromide (EtBr) staineddye

24 Evaluation of SaccharumEST-SSR across the Taxa throughCross Transferability The cross transferability of Saccharumderived EST-SSR markers was evaluated among the 20accessions comprising seven cereals (wheat maize barleyrice pearl millet oat and Sorghum) four Saccharum relatedgenera (Erianthus Miscanthus Narenga and Sclerostachya)three Saccharum species (51NG56 (S robustum) N58 (Sspontaneum) and two clones of S officinarum (BandjermasinHitam and Gunjera)) and five Saccharum commercial culti-vars (CoS 88230 CoS 92423 UP 9530 CoS 8436 and CoS91230) All genotypes were collected from the SugarcaneResearch Institute Farm UPCSR Shahjahanpur India Fur-thermore genomic DNA from young juvenile disease-freeimmature leaves was isolated for each genotype using CTAB(cetyl trimethylammonium bromide) method [16] IsolatedDNA samples were treated with RNAase for 1 h at 37∘C andpurified by phenol extraction (25 phenol 24 chloroform 1isoamyl alcohol vvv) followed by ethanol precipitation [17]and stored at minus80∘C DNAwas quantified on 08 agarose geland the working concentration of 25 ng120583L was obtained bymaking final adjustment in 10mM TE buffer

25 Genetic Diversity Analysis The assessment of EST-SSRsin genetic diversity analysis was done among 20 plantsbelonging to distinct groups comprising cereals Saccharumrelated genera Saccharum species and Saccharum cultivarsThe allelic data of 63 EST-SSR primers were used to ascertainthe genetic relationships between 20 genotypes by clusteringanalysis Amplified bands were scored as binary data in theform of present (1) or absent (0) Dendrogram was con-structed by neighbour-joining and Jaccardrsquos algorithm using

Genetics Research International 3

FreeTree and TreeView software [18 19] The polymorphicinformation content (PIC) values were calculated for eachprimer by using the online resource of PIC Calculator (httpwwwlivacuksimkempsjpichtml)

26 Informative Assessment of Functional EST-SSR Markersbetween Bulks Plantmaterials were used as F2mapping pop-ulation comprising 209 genotypes of the sugarcane cultivarswhichwere developed from cross betweenCoS 91230 (ParentCoS 775 times Co 1148) with CoS 8436 (Parent MS 6847 timesCo 1148) from September to March (2010-2011) Groupingof genotypes was done according to their stem diameter(contrasting high and low stem diameter genotypes) intotwo sets DNA extractions were carried out from both setsand equal quantities of genomic DNA from 10 extreme highstem diameter and 10 extreme low stem diameter genotypeswere pooled into two bulks PCR amplification was donein both bulks with newly developed EST-SSR primers forinformativemarkers identifications through bulk segregationanalysis (BSA) [20]

3 Results and Discussion

31 Mining of Microsatellites in EST Sequences and SSRsCharacterization Total 10000 EST sequences related toSaccharum spp were examined from NCBI for the simplesequence repeat (SSR) identification and characterizationusing computational approach Prior to the marker decipher-ing sequence assembly was performed and 6201 (4201 kb)nonredundant sequences were detected comprising 1752contigs and 4449 singlets wherein 406 SSRs were identifiedwith 360 perfect SSRs and 37 sequences containing morethan 1 SSR and 30 SSRs in compound formation There-fore computational and experimental approach to ascertainmicrosatellites in EST libraries from public database (NCBI)turned to be very cost effective and reduces time and labourbesides expense of own libraries development EST-SSRs area more preferable DNA marker in the variety of geneticanalysis and found to be more conserved as present in thetranscribed region of the genome These were found to bemore transferable across the taxonomic boundaries and couldbe evaluated as most informative markers for variety ofgenomics applications [10 21] These are more adapted inplants comparative genetic analysis for gene identificationgene mapping marker-assisted-selection transferability andgenetic diversity [7 22ndash24] Also a variety of studies havebeen reported on sugarcane using EST-SSR markers fordesired genetic analysis [8 13 25 26]

The frequency of SSR in EST sequences was 64 includ-ing all the repeats except mononucleotide repeats This resultis comparatively higher compared to previous studies onsugarcane [8 27ndash29] Contrary to this Singh et al [13]reported higher frequency (93) in sugarcane Kumpatlaand Mukhopadhyay [30] also observed high range (265 to1062) of SSR frequency in different plant species In generalabout 5 of ESTs contained SSR which has been reportedin many plant species [31] These variations in microsatellitefrequency could be attributed to the ldquosearch criteriardquo usedtype of SSRmotif size of sequence data and the mining tools

used [24 32] In otherwords the density of themicrosatelliteswas one SSR per 1045 kb which is closely comparable toearlier studies in sugarcane with densities 1 SSR109 kb [8]and 19 kb SSR [13]

Analysis revealed that trinucleotide repeats (6674)were found to be more frequent followed by di- (2610)tetra- (467) penta- (15) and hexanucleotide (12)repeats Our observation of high frequency of trinu-cleotide repeats is in agreement with previous reports onsugarcane [8 13 27ndash29 33] Several other studies havealso represented high frequency of trinucleotide repeatsin different plant species [24 31 34ndash36] A total of 33different types of motifs were identified of which fourbelonged to dinucleotide eight belonged to trinucleotidestwelve belonged to tetranucleotide five belonged to pen-tanucleotide and two belonged to hexanucleotide repeats(Figure 1) We observed that motifs AGCT and ATATwere more frequent in dinucleotide repeat followed bymotifs CCGCGGAGCCTGAGGCCT andACGCGT intrinucleotide repeat motif AAAGCTTT in tetranucleotiderepeats motif ACAGGCCTGT in pentanucleotide repeatsand AACACCGGTGTT in hexanucleotide repeats Thepresence of motif CCGCGG was also observed in sugarcaneby different authors [13 27] Kantety et al [37] also reportedCCGCGG motif as most abundant in wheat and SorghumSimilarly both Lawson and Zhang [38] and Da Maia etal [39] also observed abundance of motif CCGCGG indifferent member of the grass family Victoria et al [35] alsodecoded motif CCGCGG in the lower plants (C reinhardtiiand P patens) Thus this predominance of CCGCGG motiffrequency has been related to a high GC-content [5] Somemotifs which are responsible for making unusual DNAfolding structure (hairpin formed bipartite triplex formedand simple loop folding) also have effect on gene expressionsand regulationsmechanism namely CCTAGG CCGGGCGGATTC and GAATTC motifs [40 41] Moreover thepresence of trinucleotide repeats in the coding region formeda distinct group and encoded amino acid tracts within thepeptide [42] We also observed predictable twenty differenttypes of amino acids including stop codon Alanine arginineglycine proline and serine were most frequent (Figure 2)This is in agreement with previous studies that reported ondifferent plant species [11 35 43]

32 Expressed Sequence Tags Annotation and PrimersDevelopment All EST sequences having SSRswere examinedby functional annotation (blastn blastx and gene ontology)After-effect sixty-three ESTs having SSRs were successfullyidentified on the basis of their involvement in the variousmetabolic processes (Figure 3) After-effect sixty-threeEST-SSRs primer pairs were designed for polymorphicnature cross transferability bulk segregation analysis andgenetic diversity in the test plants (Table 1) These selectedEST-SSRs comprised all types of repeat motifs (excludingmononucleotide repeat) and among trinucleotide repeatsthey were highly frequent with GCTCGA TCCAGGand GGTCCA repeat motifs Similarly Sharma et al [44]also used functional annotation pipelines for the moreprominent molecular markers development related to gene


Table 1 Details of selected 63 EST-SSR primer pairs used for cross transferability genetic diversity and bulks segregation analysis

Serialnumber Type Primer sequence Annealing

temperature SSR motif PIC value 119864-value Putative identities(blastnblastx)

SYMS28 F GCGTCAGAGTGTTAAAACAAG 53 (GCT)4

081 939E minus 42 Protein transport proteinSec61 beta

SYMS28 R GTGTAGAACTGGAGCATTGAG

SYMS29 F GGGCAAGCAAGAAACCAC 52 (TCC)4

091 162E minus 24 Protein translation factorSUI1

SYMS29 R GAAGAGGTCAACCAAGAACTCSYMS30 F GCGTCAGAGTGTTAAAACAAG 53 (GCT)

4086 100E minus 21 Preprotein translocase Sec


SYMS31 F GAAGCTCCCAAGCTGCTA 53 (AGCT)3

076 200E minus 12 Predicted uncharacterizedprotein

SYMS31 R CCTACAGGAAAGATTTTAGGG

SYMS32 F GTCTCTTCTCCAGTTCTCCTT 55 (TGCG)4

084 246E minus 63Predictedactin-depolymerizingfactor

SYMS32 R GCTCAACAAATGTCTCCCTA

SYMS33 F TGCACTAACATGGTTGATGT 54 (GAAG)3

086 282E minus 90 Hypothetical proteinSORBIDRAFT 03g046450

SYMS33 R GGTGATTGTAAGGGTCATCTT

SYMS34 F GTTAATGGTGGTTCCGTTC 53 (GGC)6

088 4E minus 20 Predicted uncharacterizedprotein LOC101783547

SYMS34 R ATTATCAGCGCAGAGACATCSYMS35 F GCGTCAGAGTGTTAAAACAAG 52 (GCT)

4075 100E minus 21 Preprotein translocase


SYMS36 F GGACTGTACAAGGACGACAG 53 (GCT)4

070 114E minus 41 Protein transport proteinSec61 beta subunit

SYMS36 R TCTGCTTTCTTGGATATGGTA

SYMS37 F AAGAAGGATGCAAAGAAGAAG 54 (GAT)4


SYMS37 R AGGCTTAGTAACAGCAGGTTTSYMS38 F AAGAAGGATGCAAAGAAGAAG 56 (AGA)

4086 900E minus 37 Hypothetical protein

SYMS38 R AGGCTTAGTAACAGCAGGTTTSYMS39 F GGACTGTACAAGGACGACAG mdash (GCT)

4mdash 114E minus 41 Protein transport protein

SYMS39 R TCTGCTTTCTTGGATATGGTASYMS40 F GGACTGTACAAGGACGACAG mdash (GCT)

4mdash 125E minus 40 Preprotein translocase


SYMS41 F GGACTGTACAAGGACGACAG mdash (GCT)4

mdash 962E minus 42 Protein transport proteinSec61 beta subunit

SYMS41 R TCTGCTTTCTTGGATATGGTASYMS42 F CCAAAGAGATCTTGCAGACTA mdash (ATG)

4mdash 178E minus 53 Jasmonate-induced protein

SYMS42 R CCCAACACAACAACCAATSYMS43 F CCACACAAGCAAGAAATAAAC mdash (GGT)

4mdash 857E minus 74 Dirigent-like protein

SYMS43 R TCGAACACTATGGTAAAGGTG


mdash 115E minus 41 Homeodomain-liketranscription factor

SYMS44 R TCTGCTTTCTTGGATATGGTASYMS45 F GCGTCAGAGTGTTAAAACAAG 53 (GCT)

4069 986E minus 42 Protein transport protein


Table 1 Continued



SYMS45 R GACTCTGCTTTCTTGGATATG

SYMS46 F AGCTATCTTTAGTGGGGACAT 52 (CGT)4


SYMS46 R GAGGTCTCATCGGAGCTTASYMS47 F AGGTCGTTTTAATTCCTTCC 53 (GTTTT)


SYMS47 R CGTAAATATGAACGAGGTCAGSYMS48 F AGGTCGTTTTAATTCCTTCC 53 (TTTA)

6090 400E minus 20 TPA hypothetical protein

SYMS48 R CGTAAATATGAACGAGGTCAG


mdash 115E minus 41Zinc finger A20 and AN1domains-containingprotein


SYMS50 F TCCAAGGATTTAGCTATGGAT mdash (TGT)10

mdash 679E minus 13 TPA seed maturationprotein

SYMS50 R TTCAACTACACCCTTCTGTTGSYMS51 F GCGTCAGAGTGTTAAAACAAG mdash (GCT)

4mdash 122E minus 41 Hypothetical protein

SYMS51 R ATTGTCACTTGCTATCCATTT

SYMS52 F CACCTTCTTTCCTTCTCCTC mdash (CGC)4

mdash 332E minus 47 V-type proton ATPase16 kDa proteolipid subunit

SYMS52 R GTAGATACCGAGCACACCAG

SYMS53 F TCAGTTCAGGGATGACAATAG 56 (CCGTGG)3

087 259E minus 78Homeodomain-liketranscription factorsuperfamily protein

SYMS53 R GGATAGACTGAAATCTGCTCA

SYMS54 F CAACTCGACTCTTTTCTCTCA mdash (CTC)5

mdash 413E minus 08 Protein transport proteinSEC31

SYMS54 R GGAGGTGGAACTTCCTGA


mdash 112E minus 41Protein transport proteinSec61 subunit beta-likeisoform




SYMS56 R TCTGCTTTCTTGGATATGGTASYMS57 F AAACGATCAGATACCGTTGTA mdash (CG)

6mdash 784E minus 27 Caltractin

SYMS57 R ATCAAAGAGATCAAAGGCTTC

SYMS58 F CATTTCGAAGCTCCTCCT 52 (CCTCCG)6

074 597E minus 66Zinc finger A20 and AN1domains-containingprotein

SYMS58 R TAGGCTGCACAACAATAGTCT

SYMS59 F CTCCCCCATTTCTCTTCC 53 (GCAGCC)6

080 402E minus 65 Predicted reticulon-likeprotein B1

SYMS59 R CAAGTACTCCAGCAGAGATGT

SYMS60 F CTTTTCCCTCTTCCTCTCTC mdash (CCG)5

mdash 124E minus 45 Predicted uncharacterizedtRNA-binding protein

SYMS60 R TGTCACTAACACGAATCACAA

SYMS61 F CCCTCTCCCTGCTCTTTC 54 (TCC)5

079 414E minus 57 Actin-depolymerizingfactor 3


Table 1 Continued



SYMS61 R CAGTCACAAAGTCGAAATCAT

SYMS62 F ACAACTCTTCAGTCTTCACGA 54 (CAAC)3

085 440E minus 66 Truncated alcoholdehydrogenase

SYMS62 R CCAATCTTGACATCCTTGAC

SYMS63 F GCACGGTGAAGTTCTAGTTC 54 (TCGAT)4


SYMS63 R CAGCTTCACTCATGAATTTTT



SYMS64 R TCTGCTTTCTTGGATATGGTASYMS65 F AACACAAGCAAGAAATAAACG 53 (GGT)

4051 342E minus 74 Dirigent-like protein

SYMS65 R AACACTATGGTCAAGGTGGTA


058 101E minus 41Protein transport proteinSec61 subunit beta-likeisoform

SYMS66 R GAAATCGCTCTATAAGGTTCC

SYMS67 F TCTCTCTGAAGATGATGCTTT 52 (AAG)5


SYMS67 R GTTAAGAGGCTTCCAAAGAAC

SYMS68 F CAGCTCGTCGTCTTCTTTT mdash (GTC)5

200E minus 55Putativeubiquitin-conjugatingenzyme family

SYMS68 R GTGGCTTGTTTGGATATTCTT



SYMS69 R CGTCAGACGTACTGAAATGTTSYMS70 F AACACAAGCAAGAAATAAACG 53 (GGT)

4077 158E minus 73 Putative dirigent protein










SYMS73 R TCTGCTTTCTTGGATATGGTASYMS74 F GGACTGTACAAGGACGACAG 52 (GCT)



SYMS75 F GCACCCCCAATTCGAACG 52 (ACG)3

093 178E minus 68 TPA general regulatoryfactor 1

SYMS75 R CGGTAGTCCTTGATGAGTGT




SYMS77 F CACGCAACGCAAGCACAG 55 (CCAT)3



Table 1 Continued



SYMS77 R AAGTTGATTCACCCTCATTCT

SYMS78 F CACGCAACGCAAGCACAG 53 (CGATC)3

092 104E minus 41Translocon-associatedprotein alpha subunitprecursor





SYMS80 F CTTGATCCTTGACAAAAGAGA 52 (AG)6

087 225E minus 59Predictedubiquitin-conjugatingenzyme E2

SYMS80 R ATTGCTGTTGATATTTGGATG




SYMS82 F TATCAACAAGCCTTCCATTC 53 (GTG)4

090 112E minus 30 Glycine-rich RNA-bindingprotein 2

SYMS82 R GGCTATAGTCACCACGGTAG

SYMS83 F CGACAGGGAGAAGAGTACAG 55 (GCT)4





SYMS84 R AATCGCTCTATAAGGTTCCTC

SYMS85 F CTCTTCTTCACCAATTCCTCT mdash (CCG)6


SYMS85 R CAAACCTCATAAAGAGTGCAG


093 116E minus 28 TPA translation initiationfactor 1

SYMS86 R CGTACATGAACGTAGTCCTTT

SYMS87 F GCGTCAGAGTGTTAAAACAAG mdash (GCT)4



SYMS88 F TTATAAGGAAATCCCCCACT mdash (GCC)4

mdash 771E minus 55 Hypothetical proteinSORBIDRAFT 09g000970

SYMS88 R CACCAAGTACTCATCCATCAT

SYMS89 F CATCTCCTGCTAACAATTCAC 55 (TGC)4

091 964E minus 60 Predicted NACdomain-containing protein

SYMS89 R ATTTATAGGTTGGCACCAGAG





020406080

100120140

ACG

TAG

CT

ATA

TCG

CG

AAC

GTT

AAG

CTT

AAT

ATT

ACC

GG

TAC

GC

GT

ACT

AGT

AGC

CTG

AGG

CCT

ATC

ATG

CCG

CG

GA

AAG

CTT

TA

AAT

ATT

TA

AGG

CCT

TA

ATG

ATT

CA

ATT

AAT

TAC

GC

CGTG

ACTC

AG

TGAG

CCC

TGG

AGCG

CG

CTAG

GC

CCTG

AGG

GC

CCT

CCG

GC

CGG

AA

AAG

CTT

TTA

ATAT

ATA

TTAC

AGG

CCT

GT

ACCT

CAG

GTG

AGCG

GC

CGCT

AAC

ACC

GG

TGTT

AGCA

GG

CCT

GCT

Types of motifs

Freq

uenc

y

Figure 1 Details of 33 different types of nucleotide repeat motifs belonging to di- tri- tetra- penta- and hexanucleotide repeat motifs withsequence complementarity

010203040506070

Ala

Arg

Asn Asp Cy

s

Gln

Glu Gly His Ile Leu

Lys

Met

Phe

Pro

Val

Types of amino acids

Freq

uenc

y

Ser

Stop Ty

r

Thr

Figure 2 Details of different types of predicted amino acids encoded by trinucleotide repeat motifs

transcripts Selected EST-SSRs were associated with variouspathways of metabolic process namely GO0006281 DNArepair GO0006301 postreplication repair GO0016070RNA metabolic process GO0016070 RNA metabolicprocess GO0006446 regulation of translational initiationGO0015991 ATP hydrolysis coupled proton transportGO0006629 lipid metabolic process GO0015031 proteintransport GO0005667 transcription factor complexGO0005815 microtubule organizing centre GO0003743translation initiation factor activity GO0017005 31015840-tyrosyl-DNA phosphodiesterase activity GO0030042 actinfilament depolymerization and GO0015078 hydrogen iontransmembrane transporter activity and so forth (see thecomplete details of the most promising hits of gene ontologyof EST-SSRs in the supplementary table available online athttpdxdoiorg10115520167052323)

33 Assessment of EST-SSR Marker in Selected Plants A setof 63 EST-SSR primers were evaluated for PCR optimizationpolymorphism and cross amplification in twenty genotypesbelonging to cereals plants and Saccharum related genera andSaccharum species and their commercial cultivars of which42 EST-SSR primers produced successful amplifications withboth expected and unexpected sizes (Figure 4) Among 42EST-SSRs twenty-eight belonged to trinucleotide repeatswith then seven of tetra- three of penta- three of hexa- andone of dinucleotide repeats Meanwhile PCR amplifications

produced 519 alleles (expected size) at 180 loci with an averageof 288 alleles per locusThis result is comparable with earlierstudies that reported on various plant species namely 279alleleslocus in rice varieties [45] 29 to 60 alleles per locusin maize [46] and 304 alleleslocus in rye [47] Howeverour result of alleles per locus is lower compared to previousstudies that reported on sugarcane that is 604 alleleslocus[28] 755 alleleslocus [29] and 60 alleleslocus [48] Thepolymorphic information content (PIC) was extended from051 to 093 with an average of 083 It could be encompassedthat low and high range of allelic amplifications with EST-SSRs correspond to marker polymorphism and low levelof polymorphism from EST-SSRs might be due to possibleselection against alterations in the conserved sequences ofEST-SSRs [49 50]

34 Cross Transferability The potentials of EST-SSR primerswere examined for cross transferability among 20 plantspecies belonging to cereals and Saccharum related generaand Saccharum species and their cultivars under the samePCR conditions However 42 EST-SSRs showed successfulamplifications among all the selected plants The cross trans-ferability was estimated to be 2722 in wheat 2722 inmaize 4722 in barely 4666 in rice 3611 in pearl millet5555 in oat 2611 in Sorghum 8833 inNarenga 9888in Sclerostachya 7111 in Erianthus 600 in Miscanthus7333 in Bandjermasin Hitam 5555 inGunjera 7555 in


Primary metabolic process

Cellular metabolic process

Response to stress

Macromolecule metabolic process

Cellular response to stimulus

Transport

Response to other organisms

Response to biotic stimulus

Establishment of protein localization

Response to abiotic stimulus

Immune response

Response to chemical stimulus

Regulation of biological process

Cellular component organizationCellular component organization or

biogenesis at cellular levelNitrogen compound metabolic process

Small molecule metabolic process

Catabolic process

Oxidation-reduction process

Establishment of localization in cell

Biosynthetic process

Anatomical structure morphogenesis

Actin filament-based process

Cell cycle process

Vesicle-mediated transport

Post-embryonic development

Interspecies interaction between organisms

Developmental process involved in reproduction

Cellular developmental process

Activation of immune response

Cell growth

Regulation of biological quality

Immune effector processModification of morphology or

physiology of other organismSecondary metabolic process

Embryo development

Cellular localization

Seed germination

Multicellular organismal reproductive processAnatomical structure formation

involved in morphogenesisTransmembrane transport

Dormancy process

Cellular component movement

Cell division

Response to endogenous stimulus

01 02 03 04 05 06 07 08 09 1000

00 01 02 03 04 05 06 07 08 09 10

Biological_process of GO Iv3

(a)

Figure 3 Continued


00 01 02 03 04 05 06 07 08 09 10

Cellular_component of GO Iv3

090807 1000 0604030201 05

External encapsulating structureEnvelope

Vesicle Cell-cell junction

Membrane part Ribonucleoprotein complex

Protein complex Organelle membrane

Non-membrane-bounded organelle Intracellular organelle part

Membrane Membrane-bounded organelle

Intracellular organelle Intracellular part

(b)

0807 09 1005 06030201 0400

00 01 02 03 04 05 06 07 08 09 10

Molecular_function of GO Iv3

transcription factor activitySequence-specific DNA binding

Transmembrane transporter activityTetrapyrrole binding

Oxidoreductase activity Ligase activity

Nucleotide binding Hydrolase activity

Ion binding Protein binding

Nucleic acid binding Substrate-specific transporter activity

(c)

Figure 3 Most promising results of gene ontology (GO) as horizontal bar graphs These graphs represent the distribution of GO termscategorized as a biological process (a) cellular component (b) and molecular function (c)

51NG56 550 in N58 5056 in CoS 92423 5888 in CoS88230 5111 in UP 9530 5278 in CoS 91230 and 600in CoS 8436 Meanwhile the frequency distributions of crosstransferability of EST-SSRs ranged from 2611 for Sorghumto 9888 for Sclerostachya with an average of 5586(Table 2) Saccharum related genera (7958) and Saccharumspecies (6486) showed high rate of cross transferabilitycompared to other groupsThis is in agreement with previousstudies reported on Saccharum species and Saccharum relatedgenera [12 13 51] Several earlier studies related to crosstransferability have been reported on distinct plant groupsfrom different families using EST-SSRs markers [7 52 53]This suggests that transferring ability of genic markers makesit compatible to determine genetic studies across the taxa forutilization in mapping of genes from related species alongwith genera and identification of suspended hybridizationThis can also aid vigilance of the introgression of geneticentity from wild relatives to cultivated comparative mappingand establishing evolutionary relationship between themThus microsatellites derived from expressed region of the

genome are expected to be more conserved and more trans-ferable across taxa

35 Genetic Diversity Analysis by EST-SSRs In order toevaluate the potential of EST-SSRs the genetic analysis wasdone among 20 genotypes belonging to 7 cereals (wheatmaize barley rice pearl millet oat and Sorghum) 4 Sac-charum related genera (Erianthus Miscanthus Narenga andSclerostachya) 3 Saccharum species (51NG56 (S robustum)N58 (S spontaneum) and two of S officinarum clones(Gunjera and Bandjermasin Hitam)) and 5 sugarcane com-mercial cultivars (CoS 8436 CoS 91230 CoS 88230 UP 9530and CoS 92423) The generated allelic data were used forgenetic relationships analysis by making dendrogram basedon Jaccardrsquos and neighbour-joining algorithm using FreeTreeand TreeView softwareThe dendrogram fell into three majorclusters with several edges cluster I with eight genotypescomprising most of Saccharum species and their commercialcultivars cluster II encompassing six genotypes of most ofcereals species and cluster III with six species comprising

Genetics Research International 11Ta

ble2Detailsof

crosstransferabilityof

42ES

T-SSRmarkersin

twentygeno

typesb

elon

ging

tocerealsa

ndSaccharum

related

genera

andSaccharum

speciesa

ndtheirc

ultiv

arsLanes1

to20

representn

umbero

fbands

prod

uced

inwheatm

aizebarleyric

epearlm

illetoatSorghum

NarengaScle

rosta

chyaEria

nthu

sMiscanthusB

andjermasin

Hita

mG

unjer

a51NG56N

58

CoS

92423CoS

88230UP9530C

oS91230andCoS

8436respectively

Snu

mberlane

12

34

56

78

910

1112

1314

1516

1718

1920

Prim

erpo

lymorph

ism(

)SY

284

56

12

30

25

53

23

23

31

32

395

SY29

63

59

312

35

126

77

87

76

76

68

100

SY30

10

40

07

05

66

33

43

23

23

23

80SY

310

00

00

00

23

21

12

22

02

20

050

SY32

10

01

02

02

43

13

00

10

00

00

55SY

330

02

23

53

76

46

55

52

30

23

385

SY34

00

00

00

00

00

03

25

45

55

99

85SY

350

00

10

40

52

10

01

11

22

12

165

SY36

00

00

00

00

30

05

22

20

20

02

35SY

379

75

66

55

912

1011

88

88

67

65

6100

SY38

00

03

02

07

81

00

22

12

00

30

50SY

450

00

00

20

51

01

21

10

00

00

035

SY46

01

44

10

04

40

10

00

00

00

00

35SY

470

13

32

00

10

00

00

00

00

00

025

SY48

00

00

53

08

120

53

32

13

22

00

60SY

530

00

11

33

129

21

12

21

32

13

185

SY58

00

01

10

32

10

00

00

00

00

00

25SY

590

00

00

00

40

33

00

10

00

00

020

SY61

03

75

40

34

56

34

15

35

55

55

90SY

620

00

10

20

45

32

20

00

00

00

035

SY63

00

00

00

01

14

16

11

10

10

21

55SY

650

00

00

00

00

11

10

12

32

22

150

SY66

00

00

00

01

00

03

12

00

32

21

40SY

671

25

63

30

25

63

20

02

00

00

060

SY69

15

20

10

03

12

11

11

12

10

32

80SY

701

00

10

10

52

00

10

02

03

30

045

SY72

10

20

00

01

45

08

58

13

64

68

70SY

730

02

01

20

22

01

31

41

03

00

360

SY74

21

10

11

31

32

14

15

10

30

02

80SY

751

02

33

00

06

39

76

76

04

60

065

SY76

00

00

02

01

21

40

00

10

00

00

30SY

772

56

68

43

96

93

04

43

43

02

085

SY78

22

56

46

45

76

410

910

32

87

59

100

SY79

00

00

10

03

52

05

17

72

10

06

55SY

801

10

40

10

45

62

54

55

35

45

585

SY81

00

01

26

06

68

73

34

33

33

32

80SY

820

010

31

43

91

16

54

35

34

43

290

SY83

12

06

23

42

72

13

23

13

32

33

95

SY84

00

01

23

00

20

03

31

03

33

33

60


Table2Con

tinued

Snu

mberlane

12

34

56

78

910

1112

1314

1516

1718

1920

Prim

erpo

lymorph

ism(

)SY

861

56

32

54

58

116

53

115

127

67

10100

SY89

96

54

17

37

43

104

45

73

47

35

100

SY90

50

32

52

34

34

04

36

44

23

64

90Av

erageo

ftransfe

rability

2722

2722

4722

4667

3611

5556

2611

8833

9889

7111

600

7333

5556

7556

550

5056

5889

5111

5278

600


2019181716151413121110987654321M

50bp

100 bp

200 bp300bp400bp

600bp1050bp1350bp

Figure 4The gel represents PCR amplification profile with SYMS37primer among twenty different plant species Lanes 1wheat 2maize3 barley 4 rice 5 pearl millet 6 oat 7 Sorghum 8 Narenga 9Schlerostachya 10 Erianthus 11Miscanthus 12 Bandjermasin Hitam13 Gunjera 14 51NG56 15 N58 16 CoS 92423 17 CoS 88230 18 UP9530 19 CoS 91230 and 20 CoS 8436

CoS 92423

51NG56

Bandjermasin Hitam

GunjeraCoS 88230UP 9530

CoS 8436CoS 91230

BarleyRice

Maize

Wheat

Pearl milletSorghum Erianthus

NarengaMiscanthus

N58

Oat

Sclerostachya

III

III

Figure 5Dendrogram is constructed based on allelic data producedfrom 42 EST-SSR markers using FreeTree and TreeView software

most of the Saccharum related genera along with someinterventions (Figure 5) This relationship is in agreementwith previous studies reported by other authors [12 13 51 54]Our EST-SSRs markers showed close syntenic relationshipand their evolutionary nature among the 20 genotypesinto three major clusters with some genotypes divergenceThese relationships have resulted from the expansion andcontraction of SSRs in conserved EST sequences within thesame group of plant species along with some variation havingresulted from higher evolutionary divergence among themSeveral earlier studies also reported on genetic diversityanalysis within and across the plant taxa using molecularmarker [7 8 24 48 52 55ndash57] Thus microsatellite markersdistinguished all the genotypes to certain extent and alsoprovided the realistic estimate of genetic diversity amongthem

36 Bulk Segregation Analysis (BSA) in Sugarcane All the42 EST-SSR markers were evaluated in pooled DNA bulks

SYMS69SYMS66SYMS53SYMS45SYMS30


Figure 6 The gel represents polymorphism and discriminationbetween bulks of pooled DNA with contrasting high and low plantdiameter through bulk segregation analysis

of contrasting trait of sugarcane cultivars (CoS 91230 (CoS775 times Co 1148) cross with CoS 8436 (MS 6847 times Co 1148))for the identification of reporter EST-SSR markers basedon their allelic differences between them Interestingly 10markers showed polymorphic nature and apparently dis-criminating potential between bulks through bulk segrega-tion analysis (Figure 6) Among these markers SYMS30SYMS53 SYMS82 and SYMS89 showed a better response todiscriminating the bulks BSA is the strategy that involvesthe identification of genetic markers associated with char-acter or trait which are based on their allelic differencesbetween bulks [20] Earlier studies have been establishedin sugarcane for the most prominent molecular markersdetection linked to desirable traits throughBSA For examplemolecular markers apparently linked to high fiber content inSaccharum species [58ndash60] and molecular markers used forQTL analysis and utilized for generating geneticmaps aroundresistance genes in sugarcane against diseases and peststhrough BSA [12 61 62] Several other studies also reportedon selection of different agronomic traits in sugarcane forbreeding programme with the development of molecularmarkers through BSA [1 63ndash65] Alternatively BSA approachhas been recently used for various purposes against theidentification of differential expressed gene associated withboth qualitative and quantitative using of the cDNA-AFLPapproach [66ndash69] Thus BSA approach provides the easiestway in the direction of trait linked marker identification andalso makes it possible to select informative markers besideevaluations of each marker in the whole progeny


4 Conclusion

The present study was intended for identification and char-acterization of SSR in Saccharum spp expressed sequencetag which is retrieved from public database (NCBI) Furtherfunctional annotation was feasible to identify the most emi-nent EST-SSR markers selectionTherefore this is the bypassway for EST-SSR markers development which reduces costand time and provides an efficient way to analyze the tran-scribed portion of genome besides expense of own librariesdevelopment A total of 63 EST-SSR markers were developedand experimentally validated for cross transferability alongwith their genetic relationships and also used for differen-tiation between pooled DNA bulks of Saccharum cultivarsThese markers showed successful transferability rate amongthe twenty genotypes and established genetic diversity amongcereals Saccharum speciescultivars and Saccharum relatedgenera with some inconsistency Further some prominentmarker also distinguished pooled DNA bulks of sugarcanecultivars based on stem diameter Consequently these EST-SSR markers were found to be more convenient which madeit easy for us to use them as informative markers in furthergenetic studies in sugarcane breeding programme

Competing Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Authors are highly grateful to the Division of Biotechnol-ogy UP Council of Sugarcane Research for providing anopportunity and facilities for research works Authors arealso grateful to Director UP Council of Sugarcane ResearchShahjahanpur UP India for their moral support Authorsalso acknowledgeUniversity of Rajasthan for providingDBT-IPLS and DBT-BIF facilities

References

[1] J Y Hoarau G Souza A DrsquoHont et al Sugarcane A TropicalCrop with a Highly Complex Genome Plant Genomics SciencesPublishers Enfield NH USA 2006

[2] B Roach and J Daniels ldquoA review of the origin and improve-ment of sugarcanerdquo Copersucar International Sugarcane Breed-ing Workshop vol 1 pp 1ndash31 1987

[3] A DrsquoHont D Ison K Alix C Roux and J C GlaszmannldquoDetermination of basic chromosome numbers in the genusSaccharum by physical mapping of ribosomal RNA genesrdquoGenome vol 41 no 2 pp 221ndash225 1998

[4] R E Casu J M Manners G D Bonnett et al ldquoGenomicsapproaches for the identification of genes determining impor-tant traits in sugarcanerdquo Field Crops Research vol 92 no 2-3pp 137ndash147 2005

[5] MMorgante andAMOlivieri ldquoPCR-amplifiedmicrosatellitesas markers in plant geneticsrdquo Plant Journal vol 3 no 1 pp 175ndash182 1993

[6] M Agarwal N Shrivastava andH Padh ldquoAdvances inmolecu-lar marker techniques and their applications in plant sciencesrdquoPlant Cell Reports vol 27 no 4 pp 617ndash631 2008

[7] S U Haq R Jain M Sharma S Kachhwaha and S LKothari ldquoIdentification and characterization of microsatellitesin expressed sequence tags and their cross transferability indifferent plantsrdquo International Journal of Genomics vol 2014Article ID 863948 12 pages 2014

[8] S K Parida A Pandit K Gaikwad et al ldquoFunctionally relevantmicrosatellites in sugarcane unigenesrdquo BMC Plant Biology vol10 no 1 article 251 2010

[9] X Li W Gao H Guo X Zhang D D Fang and Z LinldquoDevelopment of EST-based SNP and InDel markers andtheir utilization in tetraploid cotton genetic mappingrdquo BMCGenomics vol 15 no 1 article 1046 2014

[10] Q Kong C Xiang Z Yu et al ldquoMining and characteringmicrosatellites in Cucumis melo expressed sequence tags fromsequence databaserdquo Molecular Ecology Notes vol 7 no 2 pp281ndash283 2007

[11] S Gupta K P Tripathi S Roy and A Sharma ldquoAnalysis ofunigene derived microsatellite markers in family solanaceaerdquoBioinformation vol 5 no 3 pp 113ndash121 2010

[12] R K Singh R B Singh S P Singh and M L Sharma ldquoIdenti-fication of sugarcane microsatellites associated to sugar contentin sugarcane and transferability to other cereal genomesrdquoEuphytica vol 182 no 3 pp 335ndash354 2011

[13] R K Singh S N Jena S Khan et al ldquoDevelopment cross-speciesgenera transferability of novel EST-SSR markers andtheir utility in revealing population structure and geneticdiversity in sugarcanerdquo Gene vol 524 no 2 pp 309ndash329 2013

[14] F M You N Huo Y Q Gu et al ldquoBatchPrimer3 a highthroughput web application for PCR and sequencing primerdesignrdquo BMC Bioinformatics vol 9 article 253 2008

[15] T-W Chen R-C R Gan T H Wu et al ldquoFastAnnotatormdashanefficient transcript annotationweb toolrdquoBMCGenomics vol 13no 7 article S9 2012

[16] D Hoisington M Khairallah and D Gonzalez-de-Leon Labo-ratory Protocols CIMMYT Applied Molecular Genetics Labora-tory CIMMYT Mexico DF Mexico 2nd edition 1994

[17] J Sambrook andDW RussellMolecular Cloning A LaboratoryManual Cold Spring Harbor Laboratory Press Cold SpringHarbor New York NY USA 2001

[18] A Pavlıcek S Hrda and J Flegr ldquoFree-treemdashfreeware programfor construction of phylogenetic trees on the basis of distancedata and bootstrapjackknife analysis of the tree robustnessApplication in the RAPD analysis of genus Frenkeliardquo FoliaBiologica vol 45 no 3 pp 97ndash99 1999

[19] R D Page ldquoTreeView an application to display phylogenetictrees on personal computersrdquo Computer Applications in theBiosciences vol 12 no 4 pp 357ndash358 1996

[20] R W Mlchelmore I Paran and R V Kesseli ldquoIdentification ofmarkers linked to disease-resistance genes by bulked segregantanalysis a rapid method to detect markers in specific genomicregions by using segregating populationsrdquo Proceedings of theNational Academy of Sciences of the United States of Americavol 88 no 21 pp 9828ndash9832 1991

[21] J R Ellis and J M Burke ldquoEST-SSRs as a resource forpopulation genetic analysesrdquo Heredity vol 99 no 2 pp 125ndash132 2007

[22] J Squirrell P M Hollingsworth M Woodhead et al ldquoHowmuch effort is required to isolate nuclear microsatellites fromplantsrdquoMolecular Ecology vol 12 no 6 pp 1339ndash1348 2003


[23] C Duran N Appleby D Edwards and J Batley ldquoMoleculargenetic markers discovery applications data storage and visu-alisationrdquo Current Bioinformatics vol 4 no 1 pp 16ndash27 2009

[24] K Kumari M Muthamilarasan G Misra et al ldquoDevelopmentof eSSR-markers in setaria italica and their applicability instudying genetic diversity cross-transferability and compara-tive mapping in millet and non-millet speciesrdquo PLoS ONE vol8 no 6 article e67742 2013

[25] M Rossi P G Araujo F Paulet et al ldquoGenomic distributionand characterization of EST-derived resistance gene analogs(RGAs) in sugarcanerdquo Molecular Genetics and Genomics vol269 no 3 pp 406ndash419 2003

[26] K S Aitken P A Jackson and C L McIntyre ldquoA combinationof AFLP and SSRmarkers provides extensive map coverage andidentification of homo(eo)logous linkage groups in a sugarcanecultivarrdquo Theoretical and Applied Genetics vol 110 no 5 pp789ndash801 2005

[27] G M Cordeiro R Casu C L McIntyre J M Manners and RJ Henry ldquoMicrosatellite markers from sugarcane (Saccharumspp) ESTs cross transferable to erianthus and sorghumrdquo PlantScience vol 160 no 6 pp 1115ndash1123 2001

[28] L R Pinto K M Oliveira E C Ulian A A F Garcia and AP De Souza ldquoSurvey in the sugarcane expressed sequence tagdatabase (SUCEST) for simple sequence repeatsrdquo Genome vol47 no 5 pp 795ndash804 2004

[29] K M Oliveira L R Pinto T G Marconi et al ldquoCharacteri-zation of new polymorphic functional markers for sugarcanerdquoGenome vol 52 no 2 pp 191ndash209 2009

[30] S P Kumpatla and S Mukhopadhyay ldquoMining and surveyof simple sequence repeats in expressed sequence tags ofdicotyledonous speciesrdquo Genome vol 48 no 6 pp 985ndash9982005

[31] R K Varshney A Graner and M E Sorrells ldquoGenic microsat-ellite markers in plants features and applicationsrdquo Trends inBiotechnology vol 23 no 1 pp 48ndash55 2005

[32] E Portis I Nagy Z Sasvari A Stagel L Barchi and S LanterildquoThe design of Capsicum spp SSR assays via analysis of in silicoDNA sequence and their potential utility for genetic mappingrdquoPlant Science vol 172 no 3 pp 640ndash648 2007

[33] B T James C Chen A Rudolph et al ldquoDevelopment ofmicrosatellite markers in autopolyploid sugarcane and com-parative analysis of conserved microsatellites in sorghum andsugarcanerdquoMolecular Breeding vol 30 no 2 pp 661ndash669 2012

[34] L Qiu C Yang B Tian J-B Yang and A Liu ldquoExploitingESTdatabases for the development and characterization of EST-SSR markers in castor bean (Ricinus communis L)rdquo BMC PlantBiology vol 10 no 1 article 278 2010

[35] F C Victoria L C da Maia and A C de Oliveira ldquoIn silicocomparative analysis of SSR markers in plantsrdquo BMC PlantBiology vol 11 article 15 2011

[36] M Muthamilarasan B V Suresh G Pandey K Kumari SK Parida and M Prasad ldquoDevelopment of 5123 intron-lengthpolymorphicmarkers for large-scale genotyping applications infoxtail milletrdquo DNA Research vol 21 no 1 pp 41ndash52 2014

[37] R V Kantety M La Rota D E Matthews and M E Sor-rells ldquoData mining for simple sequence repeats in expressedsequence tags from barley maize rice sorghum and wheatrdquoPlant Molecular Biology vol 48 no 5-6 pp 501ndash510 2002

[38] M J Lawson and L Zhang ldquoDistinct patterns of SSR distri-bution in the Arabidopsis thaliana and rice genomesrdquo GenomeBiology vol 7 no 2 article R14 2006

[39] L C Da Maia V Q De Souza M M Kopp F I F DeCarvalho andA CDeOliveira ldquoTandem repeat distribution ofgene transcripts in three plant familiesrdquoGenetics andMolecularBiology vol 32 no 4 pp 822ndash833 2009

[40] I Fabregat K S Koch T Aoki et al ldquoFunctional pleiotropy ofan intramolecular triplex-forming fragment from the 31015840-UTRof the rat Pigr generdquo Physiological Genomics vol 5 no 2 pp53ndash65 2001

[41] Y-C Li A B Korol T Fahima A Beiles and E NevoldquoMicrosatellites genomic distribution putative functions andmutational mechanisms a reviewrdquo Molecular Ecology vol 11no 12 pp 2453ndash2465 2002

[42] J R Gatchel and H Y Zoghbi ldquoDiseases of unstable repeatexpansion mechanisms and common principlesrdquo NatureReviews Genetics vol 6 no 10 pp 743ndash755 2005

[43] R K Mishra B H Gangadhar J W Yu D H Kim and SW Park ldquoDevelopment and characterization of EST based SSRmarkers in Madagascar periwinkle (Catharanthus roseus) andtheir transferability in other medicinal plantsrdquo Plant OmicsJournal vol 4 no 3 pp 154ndash162 2011

[44] R K Sharma P Bhardwaj R Negi T Mohapatra and PS Ahuja ldquoIdentification characterization and utilization ofunigene derivedmicrosatellite markers in tea (Camellia sinensisL)rdquo BMC Plant Biology vol 9 no 1 article 53 2009

[45] V Pachauri N Taneja P Vikram N K Singh and S SinghldquoMolecular andmorphological characterization of Indian farm-ers rice varieties (Oryza sativa L)rdquo Australian Journal of CropScience vol 7 no 7 pp 923ndash932 2013

[46] B Stich A E Melchinger M Frisch H P Maurer M Heck-enberger and J C Reif ldquoLinkage disequilibrium in Europeanelite maize germplasm investigated with SSRsrdquoTheoretical andApplied Genetics vol 111 no 4 pp 723ndash730 2005

[47] B Hackauf and P Wehling ldquoIdentification of microsatellitepolymorphisms in an expressed portion of the rye genomerdquoPlant Breeding vol 121 no 1 pp 17ndash25 2002

[48] T G Marconi E A Costa H R Miranda et al ldquoFunctionalmarkers for gene mapping and genetic diversity studies insugarcanerdquo BMC Research Notes vol 4 no 1 article 264 2011

[49] M C Saha M A R Mian I Eujayl J C Zwonitzer LWang and G D May ldquoTall fescue EST-SSR markers withtransferability across several grass speciesrdquo Theoretical andApplied Genetics vol 109 no 4 pp 783ndash791 2004

[50] S Feingold J Lloyd N Norero M Bonierbale and J LorenzenldquoMapping and characterization of new EST-derived microsatel-lites for potato (Solanum tuberosum L)rdquoTheoretical andAppliedGenetics vol 111 no 3 pp 456ndash466 2005

[51] S K Parida S K Kalia S Kaul et al ldquoInformative genomicmicrosatellite markers for efficient genotyping applications insugarcanerdquo Theoretical and Applied Genetics vol 118 no 2 pp327ndash338 2009

[52] S Gupta and M Prasad ldquoDevelopment and characterization ofgenic SSR markers in Medicago truncatula and their transfer-ability in leguminous and non-leguminous speciesrdquo Genomevol 52 no 9 pp 761ndash771 2009

[53] S K Gupta R Bansal U J Vaidya and T GopalakrishnaldquoDevelopment of EST-derived microsatellite markers in mung-bean [Vigna radiata (L) Wilczek] and their transferabilityto other Vigna speciesrdquo Indian Journal of Genetics and PlantBreeding vol 72 no 4 pp 468ndash471 2012

[54] M S Khan S Yadav S Srivastava M Swapna A Chandra andR K Singh ldquoDevelopment and utilisation of conserved-intron


scanning marker in sugarcanerdquo Australian Journal of Botanyvol 59 no 1 pp 38ndash45 2011

[55] L Y Zhang M Bernard P Leroy C Feuillet and P SourdilleldquoHigh transferability of bread wheat EST-derived SSRs to othercerealsrdquoTheoretical and Applied Genetics vol 111 no 4 pp 677ndash687 2005

[56] A Selvi N V Nair J L Noyer et al ldquoAFLP analysis of thephenetic organization and genetic diversity in the sugarcanecomplex Saccharum and Erianthusrdquo Genetic Resources andCrop Evolution vol 53 no 4 pp 831ndash842 2006

[57] M Raghami A I Lopez-Sese M R Hasandokht Z ZamaniM R F Moghadam and A Kashi ldquoGenetic diversity amongmelon accessions from Iran and their relationships with melongermplasm of diverse origins using microsatellite markersrdquoPlant Systematics and Evolution vol 300 no 1 pp 139ndash151 2014

[58] B W S Sobral and R J Honeycutt ldquoHigh output genetic map-ping of polyploids using PCR-generated markersrdquo Theoreticaland Applied Genetics vol 86 no 1 pp 105ndash112 1993

[59] N Msomi and F Botha ldquoIdentification of molecular markerslinked to fibre using bulk segregant analysisrdquo in Proceedings ofthe South African Sugar Technologistsrsquo Association (SASTA rsquo94)vol 68 pp 41ndash45 1994

[60] L Nivetha N Nair P Prathima and A Selvi ldquoIdentificationof microsatellite markers for high fibre content in sugarcanerdquoJournal of Sugarcane Research vol 3 no 1 pp 14ndash19 2013

[61] C M Dussle M Quint A E Melchinger M L Xu andT Lubberstedt ldquoSaturation of two chromosome regions con-ferring resistance to SCMV with SSR and AFLP markers bytargeted BSArdquo Theoretical and Applied Genetics vol 106 no 3pp 485ndash493 2003

[62] C Asnaghi D Roques S Ruffel et al ldquoTargeted mapping ofa sugarcane rust resistance gene (Bru1) using bulked segregantanalysis and AFLP markersrdquo Theoretical and Applied Geneticsvol 108 no 4 pp 759ndash764 2004

[63] J H Daugrois L Grivet D Roques et al ldquoA putative majorgene for rust resistance linked with a RFLPmarker in sugarcanecultivar lsquoR570rsquordquo Theoretical and Applied Genetics vol 92 no 8pp 1059ndash1064 1996

[64] C T Guimaraes G R Sills and B W S Sobral ldquoComparativemapping of Andropogoneae Saccharum L (sugarcane) and itsrelation to sorghum and maizerdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 94 no26 pp 14261ndash14266 1997

[65] R Ming S-C Liu P H Moore J E Irvine and A H PatersonldquoQTL analysis in a complex autopolyploid genetic control ofsugar content in sugarcanerdquo Genome Research vol 11 no 12pp 2075ndash2084 2001

[66] J Guo R H Y Jiang L G Kamphuis and F Govers ldquoAcDNA-AFLP based strategy to identify transcripts associatedwith avirulence in Phytophthora infestansrdquo Fungal Genetics andBiology vol 43 no 2 pp 111ndash123 2006

[67] A Fernandez-del-Carmen C Celis-Gamboa R G F Visserand C W B Bachem ldquoTargeted transcript mapping for agro-nomic traits in potatordquo Journal of Experimental Botany vol 58no 11 pp 2761ndash2774 2007

[68] B KloostermanMOortwijn J uitdeWilligen et al ldquoFromQTLto candidate gene genetical genomics of simple and complextraits in potato using a pooling strategyrdquo BMC Genomics vol11 no 1 article 158 2010

[69] X Chen P E Hedley J Morris H Liu R E Niks and N RWaugh ldquoCombining genetical genomics and bulked segregant

analysis-based differential expression an approach to genelocalizationrdquoTheoretical and Applied Genetics vol 122 no 7 pp1375ndash1383 2011

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


favored for a variety of genetic applications due to theirmultiallelic nature high reproducibility cross transferabilitycodominant inheritance abundance and extensive genomecoverage [6ndash8] Microsatellites or simple sequences repeats(SSRs) are monotonous repetitions of very short (one tosix) nucleotide motifs which occur as interspersed repet-itive elements in all eukaryotic and prokaryotic genomesHowever transcribed regions of the genome also containenormous range of microsatellites that correspond to genicmicrosatellites or EST-SSRs Therefore expressed sequencetags (ESTs) are the short transcribed portions and involvedin the variety of metabolic functions The presence of themicrosatellites in genes as well as ESTs unveils the biologicalsignificance of SSR distribution expansion and contractionon the function of the genes themselves [9]

Presently huge amounts of expressed sequence tagshave been deposited in public database (NCBI) In silicoapproaches to retrieve EST sequences from NCBI and func-tional annotations provide more constructive EST-SSRs orgene-based SSR (genic SSRs) marker development besidesown EST libraries developmentThis method of the EST-SSRmarkers development provides the easiest way to reduce costtime and labours along with more meaningful marker iden-tifications [10] The presence of microsatellites in the genicregion is found to be more conserved due to which they pos-sess high reproducibility and high interspecificintraspecifictransferability Hence EST-SSR could be used for polymor-phism genetic diversity cross transferability and compara-tive mapping in different plant species Accordingly severalgenetic studies were done on sugarcane using microsatellitemarkers to decipher polymorphism cross transferabilitygenetic diversity informative marker detection through bulksegregation analysis (BSA) and comparative genomics [811ndash13] The objective of the present study was to retrieveEST sequences for more informative EST-SSR developmentand their genetic assessment within and across the taxathrough cross transferability genetic relationships and bulksegregation analysis

2 Materials and Methods

21 EST Sequences Retrieving ESTs Assembling andMicrosatellites Identification Total 10000 EST sequences ofthe Saccharum spp were downloaded in Fasta format fromNational Centre for Biotechnology Information (NCBI) formicrosatellites deciphering Further ESTs assembling wascarried out using CAP3 programme (httpmobylepasteurfrcgi-binportalpyformscap3) for minimization of se-quences redundancyMicrosatellite identificationwas carriedout using MISA software (httppgrcipk-gaterslebendemisa) and the criteria for SSR detection were 6 4 3 3and 3 repeat units for di- tri- tetra- penta- and hexanu-cleotides respectively SSR primer pairs (forward andreverse) were designed for the selected EST sequences havingmicrosatellites using online web tool batch primer 3 pipeline[14]

22 EST-SSR Sequences Annotation Assessment of ESTsequences having SSR was done through blastnblastx

analysis for homology search and against nonredundant (nr)protein at the NCBI Furthermore functional annotationpipeline was also run at online tool for gene ontology (GO)which was intended for different GO functional classeslike biological process cellular component and molecularfunction [15]

23 PCR Amplification and Electrophoresis PCR reactionswere carried out in a total of 10 120583L volume containing 25 ngtemplate DNA 10 120583L (10 pmol120583L) of each forward andreverse primer 100mM of dNTPs 05U of Taq DNA poly-merase and 10 120583L of 10x PCR buffer with 25mM of MgCl

2

Amplification was performed in a thermal cycler (Bio-Rad)in the following conditions initial denaturation at 94∘C for5min followed by 30 amplification cycles of denaturation for1min at 94∘C followed by annealing temperature (119879

119886) for

1min and then extension for 2min at 72∘C final extension at72∘C for 7min was allowed The PCR conditions particularlythe annealing temperatures (varying from 52∘C to 58∘C) foreach primer were standardized and amplified products werestored at 4∘C The PCR products were analyzed on a 7native PAGE in vertical gel electrophoresis unit (BangaloreGenei) using TBE buffer The sizes of amplified fragmentswere estimated using 50 bp DNA ladder (Fermentas) Gelswere documented using ethidium bromide (EtBr) staineddye

24 Evaluation of SaccharumEST-SSR across the Taxa throughCross Transferability The cross transferability of Saccharumderived EST-SSR markers was evaluated among the 20accessions comprising seven cereals (wheat maize barleyrice pearl millet oat and Sorghum) four Saccharum relatedgenera (Erianthus Miscanthus Narenga and Sclerostachya)three Saccharum species (51NG56 (S robustum) N58 (Sspontaneum) and two clones of S officinarum (BandjermasinHitam and Gunjera)) and five Saccharum commercial culti-vars (CoS 88230 CoS 92423 UP 9530 CoS 8436 and CoS91230) All genotypes were collected from the SugarcaneResearch Institute Farm UPCSR Shahjahanpur India Fur-thermore genomic DNA from young juvenile disease-freeimmature leaves was isolated for each genotype using CTAB(cetyl trimethylammonium bromide) method [16] IsolatedDNA samples were treated with RNAase for 1 h at 37∘C andpurified by phenol extraction (25 phenol 24 chloroform 1isoamyl alcohol vvv) followed by ethanol precipitation [17]and stored at minus80∘C DNAwas quantified on 08 agarose geland the working concentration of 25 ng120583L was obtained bymaking final adjustment in 10mM TE buffer

25 Genetic Diversity Analysis The assessment of EST-SSRsin genetic diversity analysis was done among 20 plantsbelonging to distinct groups comprising cereals Saccharumrelated genera Saccharum species and Saccharum cultivarsThe allelic data of 63 EST-SSR primers were used to ascertainthe genetic relationships between 20 genotypes by clusteringanalysis Amplified bands were scored as binary data in theform of present (1) or absent (0) Dendrogram was con-structed by neighbour-joining and Jaccardrsquos algorithm using




























































Table 1 Continued
















































Table 1 Continued

















































Table 1 Continued












































020406080

100120140

ACG

TAG

CT

ATA

TCG

CG

AAC

GTT

AAG

CTT

AAT

ATT

ACC

GG

TAC

GC

GT

ACT

AGT

AGC

CTG

AGG

CCT

ATC

ATG

CCG

CG

GA

AAG

CTT

TA

AAT

ATT

TA

AGG

CCT

TA

ATG

ATT

CA

ATT

AAT

TAC

GC

CGTG

ACTC

AG

TGAG

CCC

TGG

AGCG

CG

CTAG

GC

CCTG

AGG

GC

CCT

CCG

GC

CGG

AA

AAG

CTT

TTA

ATAT

ATA

TTAC

AGG

CCT

GT

ACCT

CAG

GTG

AGCG

GC

CGCT

AAC

ACC

GG

TGTT

AGCA

GG

CCT

GCT

Types of motifs

Freq

uenc

y


010203040506070

Ala

Arg

Asn Asp Cy

s

Gln

Glu Gly His Ile Leu

Lys

Met

Phe

Pro

Val


Freq

uenc

y

Ser

Stop Ty

r

Thr









Response to stress



Transport





Immune response






Catabolic process






Cell cycle process







Cell growth




Embryo development


Seed germination



Dormancy process


Cell division


01 02 03 04 05 06 07 08 09 1000

00 01 02 03 04 05 06 07 08 09 10


(a)

Figure 3 Continued


00 01 02 03 04 05 06 07 08 09 10


090807 1000 0604030201 05








(b)

0807 09 1005 06030201 0400

00 01 02 03 04 05 06 07 08 09 10








(c)






ble2Detailsof


42ES

T-SSRmarkersin

twentygeno

typesb

elon

ging

tocerealsa

ndSaccharum

related

genera

andSaccharum

speciesa

ndtheirc

ultiv

arsLanes1

to20

representn

umbero

fbands

prod

uced

inwheatm

aizebarleyric

epearlm

illetoatSorghum

NarengaScle

rosta

chyaEria

nthu

sMiscanthusB

andjermasin

Hita

mG

unjer

a51NG56N

58

CoS

92423CoS

88230UP9530C

oS91230andCoS

8436respectively

Snu

mberlane

12

34

56

78

910

1112

1314

1516

1718

1920

Prim

erpo

lymorph

ism(

)SY

284

56

12

30

25

53

23

23

31

32

395

SY29

63

59

312

35

126

77

87

76

76

68

100

SY30

10

40

07

05

66

33

43

23

23

23

80SY

310

00

00

00

23

21

12

22

02

20

050

SY32

10

01

02

02

43

13

00

10

00

00

55SY

330

02

23

53

76

46

55

52

30

23

385

SY34

00

00

00

00

00

03

25

45

55

99

85SY

350

00

10

40

52

10

01

11

22

12

165

SY36

00

00

00

00

30

05

22

20

20

02

35SY

379

75

66

55

912

1011

88

88

67

65

6100

SY38

00

03

02

07

81

00

22

12

00

30

50SY

450

00

00

20

51

01

21

10

00

00

035

SY46

01

44

10

04

40

10

00

00

00

00

35SY

470

13

32

00

10

00

00

00

00

00

025

SY48

00

00

53

08

120

53

32

13

22

00

60SY

530

00

11

33

129

21

12

21

32

13

185

SY58

00

01

10

32

10

00

00

00

00

00

25SY

590

00

00

00

40

33

00

10

00

00

020

SY61

03

75

40

34

56

34

15

35

55

55

90SY

620

00

10

20

45

32

20

00

00

00

035

SY63

00

00

00

01

14

16

11

10

10

21

55SY

650

00

00

00

00

11

10

12

32

22

150

SY66

00

00

00

01

00

03

12

00

32

21

40SY

671

25

63

30

25

63

20

02

00

00

060

SY69

15

20

10

03

12

11

11

12

10

32

80SY

701

00

10

10

52

00

10

02

03

30

045

SY72

10

20

00

01

45

08

58

13

64

68

70SY

730

02

01

20

22

01

31

41

03

00

360

SY74

21

10

11

31

32

14

15

10

30

02

80SY

751

02

33

00

06

39

76

76

04

60

065

SY76

00

00

02

01

21

40

00

10

00

00

30SY

772

56

68

43

96

93

04

43

43

02

085

SY78

22

56

46

45

76

410

910

32

87

59

100

SY79

00

00

10

03

52

05

17

72

10

06

55SY

801

10

40

10

45

62

54

55

35

45

585

SY81

00

01

26

06

68

73

34

33

33

32

80SY

820

010

31

43

91

16

54

35

34

43

290

SY83

12

06

23

42

72

13

23

13

32

33

95

SY84

00

01

23

00

20

03

31

03

33

33

60


Table2Con

tinued

Snu

mberlane

12

34

56

78

910

1112

1314

1516

1718

1920

Prim

erpo

lymorph

ism(

)SY

861

56

32

54

58

116

53

115

127

67

10100

SY89

96

54

17

37

43

104

45

73

47

35

100

SY90

50

32

52

34

34

04

36

44

23

64

90Av

erageo

ftransfe

rability

2722

2722

4722

4667

3611

5556

2611

8833

9889

7111

600

7333

5556

7556

550

5056

5889

5111

5278

600


2019181716151413121110987654321M

50bp

100 bp

200 bp300bp400bp

600bp1050bp1350bp


CoS 92423

51NG56

Bandjermasin Hitam


CoS 8436CoS 91230

BarleyRice

Maize

Wheat


NarengaMiscanthus

N58

Oat

Sclerostachya

III

III









4 Conclusion


Competing Interests


Acknowledgments


References

















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




























































Table 1 Continued
















































Table 1 Continued

















































Table 1 Continued












































020406080

100120140

ACG

TAG

CT

ATA

TCG

CG

AAC

GTT

AAG

CTT

AAT

ATT

ACC

GG

TAC

GC

GT

ACT

AGT

AGC

CTG

AGG

CCT

ATC

ATG

CCG

CG

GA

AAG

CTT

TA

AAT

ATT

TA

AGG

CCT

TA

ATG

ATT

CA

ATT

AAT

TAC

GC

CGTG

ACTC

AG

TGAG

CCC

TGG

AGCG

CG

CTAG

GC

CCTG

AGG

GC

CCT

CCG

GC

CGG

AA

AAG

CTT

TTA

ATAT

ATA

TTAC

AGG

CCT

GT

ACCT

CAG

GTG

AGCG

GC

CGCT

AAC

ACC

GG

TGTT

AGCA

GG

CCT

GCT

Types of motifs

Freq

uenc

y


010203040506070

Ala

Arg

Asn Asp Cy

s

Gln

Glu Gly His Ile Leu

Lys

Met

Phe

Pro

Val


Freq

uenc

y

Ser

Stop Ty

r

Thr









Response to stress



Transport





Immune response






Catabolic process






Cell cycle process







Cell growth




Embryo development


Seed germination



Dormancy process


Cell division


01 02 03 04 05 06 07 08 09 1000

00 01 02 03 04 05 06 07 08 09 10


(a)

Figure 3 Continued


00 01 02 03 04 05 06 07 08 09 10


090807 1000 0604030201 05








(b)

0807 09 1005 06030201 0400

00 01 02 03 04 05 06 07 08 09 10








(c)






ble2Detailsof


42ES

T-SSRmarkersin

twentygeno

typesb

elon

ging

tocerealsa

ndSaccharum

related

genera

andSaccharum

speciesa

ndtheirc

ultiv

arsLanes1

to20

representn

umbero

fbands

prod

uced

inwheatm

aizebarleyric

epearlm

illetoatSorghum

NarengaScle

rosta

chyaEria

nthu

sMiscanthusB

andjermasin

Hita

mG

unjer

a51NG56N

58

CoS

92423CoS

88230UP9530C

oS91230andCoS

8436respectively

Snu

mberlane

12

34

56

78

910

1112

1314

1516

1718

1920

Prim

erpo

lymorph

ism(

)SY

284

56

12

30

25

53

23

23

31

32

395

SY29

63

59

312

35

126

77

87

76

76

68

100

SY30

10

40

07

05

66

33

43

23

23

23

80SY

310

00

00

00

23

21

12

22

02

20

050

SY32

10

01

02

02

43

13

00

10

00

00

55SY

330

02

23

53

76

46

55

52

30

23

385

SY34

00

00

00

00

00

03

25

45

55

99

85SY

350

00

10

40

52

10

01

11

22

12

165

SY36

00

00

00

00

30

05

22

20

20

02

35SY

379

75

66

55

912

1011

88

88

67

65

6100

SY38

00

03

02

07

81

00

22

12

00

30

50SY

450

00

00

20

51

01

21

10

00

00

035

SY46

01

44

10

04

40

10

00

00

00

00

35SY

470

13

32

00

10

00

00

00

00

00

025

SY48

00

00

53

08

120

53

32

13

22

00

60SY

530

00

11

33

129

21

12

21

32

13

185

SY58

00

01

10

32

10

00

00

00

00

00

25SY

590

00

00

00

40

33

00

10

00

00

020

SY61

03

75

40

34

56

34

15

35

55

55

90SY

620

00

10

20

45

32

20

00

00

00

035

SY63

00

00

00

01

14

16

11

10

10

21

55SY

650

00

00

00

00

11

10

12

32

22

150

SY66

00

00

00

01

00

03

12

00

32

21

40SY

671

25

63

30

25

63

20

02

00

00

060

SY69

15

20

10

03

12

11

11

12

10

32

80SY

701

00

10

10

52

00

10

02

03

30

045

SY72

10

20

00

01

45

08

58

13

64

68

70SY

730

02

01

20

22

01

31

41

03

00

360

SY74

21

10

11

31

32

14

15

10

30

02

80SY

751

02

33

00

06

39

76

76

04

60

065

SY76

00

00

02

01

21

40

00

10

00

00

30SY

772

56

68

43

96

93

04

43

43

02

085

SY78

22

56

46

45

76

410

910

32

87

59

100

SY79

00

00

10

03

52

05

17

72

10

06

55SY

801

10

40

10

45

62

54

55

35

45

585

SY81

00

01

26

06

68

73

34

33

33

32

80SY

820

010

31

43

91

16

54

35

34

43

290

SY83

12

06

23

42

72

13

23

13

32

33

95

SY84

00

01

23

00

20

03

31

03

33

33

60


Table2Con

tinued

Snu

mberlane

12

34

56

78

910

1112

1314

1516

1718

1920

Prim

erpo

lymorph

ism(

)SY

861

56

32

54

58

116

53

115

127

67

10100

SY89

96

54

17

37

43

104

45

73

47

35

100

SY90

50

32

52

34

34

04

36

44

23

64

90Av

erageo

ftransfe

rability

2722

2722

4722

4667

3611

5556

2611

8833

9889

7111

600

7333

5556

7556

550

5056

5889

5111

5278

600


2019181716151413121110987654321M

50bp

100 bp

200 bp300bp400bp

600bp1050bp1350bp


CoS 92423

51NG56

Bandjermasin Hitam


CoS 8436CoS 91230

BarleyRice

Maize

Wheat


NarengaMiscanthus

N58

Oat

Sclerostachya

III

III









4 Conclusion


Competing Interests


Acknowledgments


References

















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table 1 Continued

















































Table 1 Continued












































020406080

100120140

ACG

TAG

CT

ATA

TCG

CG

AAC

GTT

AAG

CTT

AAT

ATT

ACC

GG

TAC

GC

GT

ACT

AGT

AGC

CTG

AGG

CCT

ATC

ATG

CCG

CG

GA

AAG

CTT

TA

AAT

ATT

TA

AGG

CCT

TA

ATG

ATT

CA

ATT

AAT

TAC

GC

CGTG

ACTC

AG

TGAG

CCC

TGG

AGCG

CG

CTAG

GC

CCTG

AGG

GC

CCT

CCG

GC

CGG

AA

AAG

CTT

TTA

ATAT

ATA

TTAC

AGG

CCT

GT

ACCT

CAG

GTG

AGCG

GC

CGCT

AAC

ACC

GG

TGTT

AGCA

GG

CCT

GCT

Types of motifs

Freq

uenc

y


010203040506070

Ala

Arg

Asn Asp Cy

s

Gln

Glu Gly His Ile Leu

Lys

Met

Phe

Pro

Val


Freq

uenc

y

Ser

Stop Ty

r

Thr









Response to stress



Transport





Immune response






Catabolic process






Cell cycle process







Cell growth




Embryo development


Seed germination



Dormancy process


Cell division


01 02 03 04 05 06 07 08 09 1000

00 01 02 03 04 05 06 07 08 09 10


(a)

Figure 3 Continued


00 01 02 03 04 05 06 07 08 09 10


090807 1000 0604030201 05








(b)

0807 09 1005 06030201 0400

00 01 02 03 04 05 06 07 08 09 10








(c)






ble2Detailsof


42ES

T-SSRmarkersin

twentygeno

typesb

elon

ging

tocerealsa

ndSaccharum

related

genera

andSaccharum

speciesa

ndtheirc

ultiv

arsLanes1

to20

representn

umbero

fbands

prod

uced

inwheatm

aizebarleyric

epearlm

illetoatSorghum

NarengaScle

rosta

chyaEria

nthu

sMiscanthusB

andjermasin

Hita

mG

unjer

a51NG56N

58

CoS

92423CoS

88230UP9530C

oS91230andCoS

8436respectively

Snu

mberlane

12

34

56

78

910

1112

1314

1516

1718

1920

Prim

erpo

lymorph

ism(

)SY

284

56

12

30

25

53

23

23

31

32

395

SY29

63

59

312

35

126

77

87

76

76

68

100

SY30

10

40

07

05

66

33

43

23

23

23

80SY

310

00

00

00

23

21

12

22

02

20

050

SY32

10

01

02

02

43

13

00

10

00

00

55SY

330

02

23

53

76

46

55

52

30

23

385

SY34

00

00

00

00

00

03

25

45

55

99

85SY

350

00

10

40

52

10

01

11

22

12

165

SY36

00

00

00

00

30

05

22

20

20

02

35SY

379

75

66

55

912

1011

88

88

67

65

6100

SY38

00

03

02

07

81

00

22

12

00

30

50SY

450

00

00

20

51

01

21

10

00

00

035

SY46

01

44

10

04

40

10

00

00

00

00

35SY

470

13

32

00

10

00

00

00

00

00

025

SY48

00

00

53

08

120

53

32

13

22

00

60SY

530

00

11

33

129

21

12

21

32

13

185

SY58

00

01

10

32

10

00

00

00

00

00

25SY

590

00

00

00

40

33

00

10

00

00

020

SY61

03

75

40

34

56

34

15

35

55

55

90SY

620

00

10

20

45

32

20

00

00

00

035

SY63

00

00

00

01

14

16

11

10

10

21

55SY

650

00

00

00

00

11

10

12

32

22

150

SY66

00

00

00

01

00

03

12

00

32

21

40SY

671

25

63

30

25

63

20

02

00

00

060

SY69

15

20

10

03

12

11

11

12

10

32

80SY

701

00

10

10

52

00

10

02

03

30

045

SY72

10

20

00

01

45

08

58

13

64

68

70SY

730

02

01

20

22

01

31

41

03

00

360

SY74

21

10

11

31

32

14

15

10

30

02

80SY

751

02

33

00

06

39

76

76

04

60

065

SY76

00

00

02

01

21

40

00

10

00

00

30SY

772

56

68

43

96

93

04

43

43

02

085

SY78

22

56

46

45

76

410

910

32

87

59

100

SY79

00

00

10

03

52

05

17

72

10

06

55SY

801

10

40

10

45

62

54

55

35

45

585

SY81

00

01

26

06

68

73

34

33

33

32

80SY

820

010

31

43

91

16

54

35

34

43

290

SY83

12

06

23

42

72

13

23

13

32

33

95

SY84

00

01

23

00

20

03

31

03

33

33

60


Table2Con

tinued

Snu

mberlane

12

34

56

78

910

1112

1314

1516

1718

1920

Prim

erpo

lymorph

ism(

)SY

861

56

32

54

58

116

53

115

127

67

10100

SY89

96

54

17

37

43

104

45

73

47

35

100

SY90

50

32

52

34

34

04

36

44

23

64

90Av

erageo

ftransfe

rability

2722

2722

4722

4667

3611

5556

2611

8833

9889

7111

600

7333

5556

7556

550

5056

5889

5111

5278

600


2019181716151413121110987654321M

50bp

100 bp

200 bp300bp400bp

600bp1050bp1350bp


CoS 92423

51NG56

Bandjermasin Hitam


CoS 8436CoS 91230

BarleyRice

Maize

Wheat


NarengaMiscanthus

N58

Oat

Sclerostachya

III

III









4 Conclusion


Competing Interests


Acknowledgments


References

















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table 1 Continued












































020406080

100120140

ACG

TAG

CT

ATA

TCG

CG

AAC

GTT

AAG

CTT

AAT

ATT

ACC

GG

TAC

GC

GT

ACT

AGT

AGC

CTG

AGG

CCT

ATC

ATG

CCG

CG

GA

AAG

CTT

TA

AAT

ATT

TA

AGG

CCT

TA

ATG

ATT

CA

ATT

AAT

TAC

GC

CGTG

ACTC

AG

TGAG

CCC

TGG

AGCG

CG

CTAG

GC

CCTG

AGG

GC

CCT

CCG

GC

CGG

AA

AAG

CTT

TTA

ATAT

ATA

TTAC

AGG

CCT

GT

ACCT

CAG

GTG

AGCG

GC

CGCT

AAC

ACC

GG

TGTT

AGCA

GG

CCT

GCT

Types of motifs

Freq

uenc

y


010203040506070

Ala

Arg

Asn Asp Cy

s

Gln

Glu Gly His Ile Leu

Lys

Met

Phe

Pro

Val


Freq

uenc

y

Ser

Stop Ty

r

Thr









Response to stress



Transport





Immune response






Catabolic process






Cell cycle process







Cell growth




Embryo development


Seed germination



Dormancy process


Cell division


01 02 03 04 05 06 07 08 09 1000

00 01 02 03 04 05 06 07 08 09 10


(a)

Figure 3 Continued


00 01 02 03 04 05 06 07 08 09 10


090807 1000 0604030201 05








(b)

0807 09 1005 06030201 0400

00 01 02 03 04 05 06 07 08 09 10








(c)






ble2Detailsof


42ES

T-SSRmarkersin

twentygeno

typesb

elon

ging

tocerealsa

ndSaccharum

related

genera

andSaccharum

speciesa

ndtheirc

ultiv

arsLanes1

to20

representn

umbero

fbands

prod

uced

inwheatm

aizebarleyric

epearlm

illetoatSorghum

NarengaScle

rosta

chyaEria

nthu

sMiscanthusB

andjermasin

Hita

mG

unjer

a51NG56N

58

CoS

92423CoS

88230UP9530C

oS91230andCoS

8436respectively

Snu

mberlane

12

34

56

78

910

1112

1314

1516

1718

1920

Prim

erpo

lymorph

ism(

)SY

284

56

12

30

25

53

23

23

31

32

395

SY29

63

59

312

35

126

77

87

76

76

68

100

SY30

10

40

07

05

66

33

43

23

23

23

80SY

310

00

00

00

23

21

12

22

02

20

050

SY32

10

01

02

02

43

13

00

10

00

00

55SY

330

02

23

53

76

46

55

52

30

23

385

SY34

00

00

00

00

00

03

25

45

55

99

85SY

350

00

10

40

52

10

01

11

22

12

165

SY36

00

00

00

00

30

05

22

20

20

02

35SY

379

75

66

55

912

1011

88

88

67

65

6100

SY38

00

03

02

07

81

00

22

12

00

30

50SY

450

00

00

20

51

01

21

10

00

00

035

SY46

01

44

10

04

40

10

00

00

00

00

35SY

470

13

32

00

10

00

00

00

00

00

025

SY48

00

00

53

08

120

53

32

13

22

00

60SY

530

00

11

33

129

21

12

21

32

13

185

SY58

00

01

10

32

10

00

00

00

00

00

25SY

590

00

00

00

40

33

00

10

00

00

020

SY61

03

75

40

34

56

34

15

35

55

55

90SY

620

00

10

20

45

32

20

00

00

00

035

SY63

00

00

00

01

14

16

11

10

10

21

55SY

650

00

00

00

00

11

10

12

32

22

150

SY66

00

00

00

01

00

03

12

00

32

21

40SY

671

25

63

30

25

63

20

02

00

00

060

SY69

15

20

10

03

12

11

11

12

10

32

80SY

701

00

10

10

52

00

10

02

03

30

045

SY72

10

20

00

01

45

08

58

13

64

68

70SY

730

02

01

20

22

01

31

41

03

00

360

SY74

21

10

11

31

32

14

15

10

30

02

80SY

751

02

33

00

06

39

76

76

04

60

065

SY76

00

00

02

01

21

40

00

10

00

00

30SY

772

56

68

43

96

93

04

43

43

02

085

SY78

22

56

46

45

76

410

910

32

87

59

100

SY79

00

00

10

03

52

05

17

72

10

06

55SY

801

10

40

10

45

62

54

55

35

45

585

SY81

00

01

26

06

68

73

34

33

33

32

80SY

820

010

31

43

91

16

54

35

34

43

290

SY83

12

06

23

42

72

13

23

13

32

33

95

SY84

00

01

23

00

20

03

31

03

33

33

60


Table2Con

tinued

Snu

mberlane

12

34

56

78

910

1112

1314

1516

1718

1920

Prim

erpo

lymorph

ism(

)SY

861

56

32

54

58

116

53

115

127

67

10100

SY89

96

54

17

37

43

104

45

73

47

35

100

SY90

50

32

52

34

34

04

36

44

23

64

90Av

erageo

ftransfe

rability

2722

2722

4722

4667

3611

5556

2611

8833

9889

7111

600

7333

5556

7556

550

5056

5889

5111

5278

600


2019181716151413121110987654321M

50bp

100 bp

200 bp300bp400bp

600bp1050bp1350bp


CoS 92423

51NG56

Bandjermasin Hitam


CoS 8436CoS 91230

BarleyRice

Maize

Wheat


NarengaMiscanthus

N58

Oat

Sclerostachya

III

III









4 Conclusion


Competing Interests


Acknowledgments


References

















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


020406080

100120140

ACG

TAG

CT

ATA

TCG

CG

AAC

GTT

AAG

CTT

AAT

ATT

ACC

GG

TAC

GC

GT

ACT

AGT

AGC

CTG

AGG

CCT

ATC

ATG

CCG

CG

GA

AAG

CTT

TA

AAT

ATT

TA

AGG

CCT

TA

ATG

ATT

CA

ATT

AAT

TAC

GC

CGTG

ACTC

AG

TGAG

CCC

TGG

AGCG

CG

CTAG

GC

CCTG

AGG

GC

CCT

CCG

GC

CGG

AA

AAG

CTT

TTA

ATAT

ATA

TTAC

AGG

CCT

GT

ACCT

CAG

GTG

AGCG

GC

CGCT

AAC

ACC

GG

TGTT

AGCA

GG

CCT

GCT

Types of motifs

Freq

uenc

y


010203040506070

Ala

Arg

Asn Asp Cy

s

Gln

Glu Gly His Ile Leu

Lys

Met

Phe

Pro

Val


Freq

uenc

y

Ser

Stop Ty

r

Thr









Response to stress



Transport





Immune response






Catabolic process






Cell cycle process







Cell growth




Embryo development


Seed germination



Dormancy process


Cell division


01 02 03 04 05 06 07 08 09 1000

00 01 02 03 04 05 06 07 08 09 10


(a)

Figure 3 Continued


00 01 02 03 04 05 06 07 08 09 10


090807 1000 0604030201 05








(b)

0807 09 1005 06030201 0400

00 01 02 03 04 05 06 07 08 09 10








(c)






ble2Detailsof


42ES

T-SSRmarkersin

twentygeno

typesb

elon

ging

tocerealsa

ndSaccharum

related

genera

andSaccharum

speciesa

ndtheirc

ultiv

arsLanes1

to20

representn

umbero

fbands

prod

uced

inwheatm

aizebarleyric

epearlm

illetoatSorghum

NarengaScle

rosta

chyaEria

nthu

sMiscanthusB

andjermasin

Hita

mG

unjer

a51NG56N

58

CoS

92423CoS

88230UP9530C

oS91230andCoS

8436respectively

Snu

mberlane

12

34

56

78

910

1112

1314

1516

1718

1920

Prim

erpo

lymorph

ism(

)SY

284

56

12

30

25

53

23

23

31

32

395

SY29

63

59

312

35

126

77

87

76

76

68

100

SY30

10

40

07

05

66

33

43

23

23

23

80SY

310

00

00

00

23

21

12

22

02

20

050

SY32

10

01

02

02

43

13

00

10

00

00

55SY

330

02

23

53

76

46

55

52

30

23

385

SY34

00

00

00

00

00

03

25

45

55

99

85SY

350

00

10

40

52

10

01

11

22

12

165

SY36

00

00

00

00

30

05

22

20

20

02

35SY

379

75

66

55

912

1011

88

88

67

65

6100

SY38

00

03

02

07

81

00

22

12

00

30

50SY

450

00

00

20

51

01

21

10

00

00

035

SY46

01

44

10

04

40

10

00

00

00

00

35SY

470

13

32

00

10

00

00

00

00

00

025

SY48

00

00

53

08

120

53

32

13

22

00

60SY

530

00

11

33

129

21

12

21

32

13

185

SY58

00

01

10

32

10

00

00

00

00

00

25SY

590

00

00

00

40

33

00

10

00

00

020

SY61

03

75

40

34

56

34

15

35

55

55

90SY

620

00

10

20

45

32

20

00

00

00

035

SY63

00

00

00

01

14

16

11

10

10

21

55SY

650

00

00

00

00

11

10

12

32

22

150

SY66

00

00

00

01

00

03

12

00

32

21

40SY

671

25

63

30

25

63

20

02

00

00

060

SY69

15

20

10

03

12

11

11

12

10

32

80SY

701

00

10

10

52

00

10

02

03

30

045

SY72

10

20

00

01

45

08

58

13

64

68

70SY

730

02

01

20

22

01

31

41

03

00

360

SY74

21

10

11

31

32

14

15

10

30

02

80SY

751

02

33

00

06

39

76

76

04

60

065

SY76

00

00

02

01

21

40

00

10

00

00

30SY

772

56

68

43

96

93

04

43

43

02

085

SY78

22

56

46

45

76

410

910

32

87

59

100

SY79

00

00

10

03

52

05

17

72

10

06

55SY

801

10

40

10

45

62

54

55

35

45

585

SY81

00

01

26

06

68

73

34

33

33

32

80SY

820

010

31

43

91

16

54

35

34

43

290

SY83

12

06

23

42

72

13

23

13

32

33

95

SY84

00

01

23

00

20

03

31

03

33

33

60


Table2Con

tinued

Snu

mberlane

12

34

56

78

910

1112

1314

1516

1718

1920

Prim

erpo

lymorph

ism(

)SY

861

56

32

54

58

116

53

115

127

67

10100

SY89

96

54

17

37

43

104

45

73

47

35

100

SY90

50

32

52

34

34

04

36

44

23

64

90Av

erageo

ftransfe

rability

2722

2722

4722

4667

3611

5556

2611

8833

9889

7111

600

7333

5556

7556

550

5056

5889

5111

5278

600


2019181716151413121110987654321M

50bp

100 bp

200 bp300bp400bp

600bp1050bp1350bp


CoS 92423

51NG56

Bandjermasin Hitam


CoS 8436CoS 91230

BarleyRice

Maize

Wheat


NarengaMiscanthus

N58

Oat

Sclerostachya

III

III









4 Conclusion


Competing Interests


Acknowledgments


References

















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




Response to stress



Transport





Immune response






Catabolic process






Cell cycle process







Cell growth




Embryo development


Seed germination



Dormancy process


Cell division


01 02 03 04 05 06 07 08 09 1000

00 01 02 03 04 05 06 07 08 09 10


(a)

Figure 3 Continued


00 01 02 03 04 05 06 07 08 09 10


090807 1000 0604030201 05








(b)

0807 09 1005 06030201 0400

00 01 02 03 04 05 06 07 08 09 10








(c)






ble2Detailsof


42ES

T-SSRmarkersin

twentygeno

typesb

elon

ging

tocerealsa

ndSaccharum

related

genera

andSaccharum

speciesa

ndtheirc

ultiv

arsLanes1

to20

representn

umbero

fbands

prod

uced

inwheatm

aizebarleyric

epearlm

illetoatSorghum

NarengaScle

rosta

chyaEria

nthu

sMiscanthusB

andjermasin

Hita

mG

unjer

a51NG56N

58

CoS

92423CoS

88230UP9530C

oS91230andCoS

8436respectively

Snu

mberlane

12

34

56

78

910

1112

1314

1516

1718

1920

Prim

erpo

lymorph

ism(

)SY

284

56

12

30

25

53

23

23

31

32

395

SY29

63

59

312

35

126

77

87

76

76

68

100

SY30

10

40

07

05

66

33

43

23

23

23

80SY

310

00

00

00

23

21

12

22

02

20

050

SY32

10

01

02

02

43

13

00

10

00

00

55SY

330

02

23

53

76

46

55

52

30

23

385

SY34

00

00

00

00

00

03

25

45

55

99

85SY

350

00

10

40

52

10

01

11

22

12

165

SY36

00

00

00

00

30

05

22

20

20

02

35SY

379

75

66

55

912

1011

88

88

67

65

6100

SY38

00

03

02

07

81

00

22

12

00

30

50SY

450

00

00

20

51

01

21

10

00

00

035

SY46

01

44

10

04

40

10

00

00

00

00

35SY

470

13

32

00

10

00

00

00

00

00

025

SY48

00

00

53

08

120

53

32

13

22

00

60SY

530

00

11

33

129

21

12

21

32

13

185

SY58

00

01

10

32

10

00

00

00

00

00

25SY

590

00

00

00

40

33

00

10

00

00

020

SY61

03

75

40

34

56

34

15

35

55

55

90SY

620

00

10

20

45

32

20

00

00

00

035

SY63

00

00

00

01

14

16

11

10

10

21

55SY

650

00

00

00

00

11

10

12

32

22

150

SY66

00

00

00

01

00

03

12

00

32

21

40SY

671

25

63

30

25

63

20

02

00

00

060

SY69

15

20

10

03

12

11

11

12

10

32

80SY

701

00

10

10

52

00

10

02

03

30

045

SY72

10

20

00

01

45

08

58

13

64

68

70SY

730

02

01

20

22

01

31

41

03

00

360

SY74

21

10

11

31

32

14

15

10

30

02

80SY

751

02

33

00

06

39

76

76

04

60

065

SY76

00

00

02

01

21

40

00

10

00

00

30SY

772

56

68

43

96

93

04

43

43

02

085

SY78

22

56

46

45

76

410

910

32

87

59

100

SY79

00

00

10

03

52

05

17

72

10

06

55SY

801

10

40

10

45

62

54

55

35

45

585

SY81

00

01

26

06

68

73

34

33

33

32

80SY

820

010

31

43

91

16

54

35

34

43

290

SY83

12

06

23

42

72

13

23

13

32

33

95

SY84

00

01

23

00

20

03

31

03

33

33

60


Table2Con

tinued

Snu

mberlane

12

34

56

78

910

1112

1314

1516

1718

1920

Prim

erpo

lymorph

ism(

)SY

861

56

32

54

58

116

53

115

127

67

10100

SY89

96

54

17

37

43

104

45

73

47

35

100

SY90

50

32

52

34

34

04

36

44

23

64

90Av

erageo

ftransfe

rability

2722

2722

4722

4667

3611

5556

2611

8833

9889

7111

600

7333

5556

7556

550

5056

5889

5111

5278

600


2019181716151413121110987654321M

50bp

100 bp

200 bp300bp400bp

600bp1050bp1350bp


CoS 92423

51NG56

Bandjermasin Hitam


CoS 8436CoS 91230

BarleyRice

Maize

Wheat


NarengaMiscanthus

N58

Oat

Sclerostachya

III

III









4 Conclusion


Competing Interests


Acknowledgments


References

















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


00 01 02 03 04 05 06 07 08 09 10


090807 1000 0604030201 05








(b)

0807 09 1005 06030201 0400

00 01 02 03 04 05 06 07 08 09 10








(c)






ble2Detailsof


42ES

T-SSRmarkersin

twentygeno

typesb

elon

ging

tocerealsa

ndSaccharum

related

genera

andSaccharum

speciesa

ndtheirc

ultiv

arsLanes1

to20

representn

umbero

fbands

prod

uced

inwheatm

aizebarleyric

epearlm

illetoatSorghum

NarengaScle

rosta

chyaEria

nthu

sMiscanthusB

andjermasin

Hita

mG

unjer

a51NG56N

58

CoS

92423CoS

88230UP9530C

oS91230andCoS

8436respectively

Snu

mberlane

12

34

56

78

910

1112

1314

1516

1718

1920

Prim

erpo

lymorph

ism(

)SY

284

56

12

30

25

53

23

23

31

32

395

SY29

63

59

312

35

126

77

87

76

76

68

100

SY30

10

40

07

05

66

33

43

23

23

23

80SY

310

00

00

00

23

21

12

22

02

20

050

SY32

10

01

02

02

43

13

00

10

00

00

55SY

330

02

23

53

76

46

55

52

30

23

385

SY34

00

00

00

00

00

03

25

45

55

99

85SY

350

00

10

40

52

10

01

11

22

12

165

SY36

00

00

00

00

30

05

22

20

20

02

35SY

379

75

66

55

912

1011

88

88

67

65

6100

SY38

00

03

02

07

81

00

22

12

00

30

50SY

450

00

00

20

51

01

21

10

00

00

035

SY46

01

44

10

04

40

10

00

00

00

00

35SY

470

13

32

00

10

00

00

00

00

00

025

SY48

00

00

53

08

120

53

32

13

22

00

60SY

530

00

11

33

129

21

12

21

32

13

185

SY58

00

01

10

32

10

00

00

00

00

00

25SY

590

00

00

00

40

33

00

10

00

00

020

SY61

03

75

40

34

56

34

15

35

55

55

90SY

620

00

10

20

45

32

20

00

00

00

035

SY63

00

00

00

01

14

16

11

10

10

21

55SY

650

00

00

00

00

11

10

12

32

22

150

SY66

00

00

00

01

00

03

12

00

32

21

40SY

671

25

63

30

25

63

20

02

00

00

060

SY69

15

20

10

03

12

11

11

12

10

32

80SY

701

00

10

10

52

00

10

02

03

30

045

SY72

10

20

00

01

45

08

58

13

64

68

70SY

730

02

01

20

22

01

31

41

03

00

360

SY74

21

10

11

31

32

14

15

10

30

02

80SY

751

02

33

00

06

39

76

76

04

60

065

SY76

00

00

02

01

21

40

00

10

00

00

30SY

772

56

68

43

96

93

04

43

43

02

085

SY78

22

56

46

45

76

410

910

32

87

59

100

SY79

00

00

10

03

52

05

17

72

10

06

55SY

801

10

40

10

45

62

54

55

35

45

585

SY81

00

01

26

06

68

73

34

33

33

32

80SY

820

010

31

43

91

16

54

35

34

43

290

SY83

12

06

23

42

72

13

23

13

32

33

95

SY84

00

01

23

00

20

03

31

03

33

33

60


Table2Con

tinued

Snu

mberlane

12

34

56

78

910

1112

1314

1516

1718

1920

Prim

erpo

lymorph

ism(

)SY

861

56

32

54

58

116

53

115

127

67

10100

SY89

96

54

17

37

43

104

45

73

47

35

100

SY90

50

32

52

34

34

04

36

44

23

64

90Av

erageo

ftransfe

rability

2722

2722

4722

4667

3611

5556

2611

8833

9889

7111

600

7333

5556

7556

550

5056

5889

5111

5278

600


2019181716151413121110987654321M

50bp

100 bp

200 bp300bp400bp

600bp1050bp1350bp


CoS 92423

51NG56

Bandjermasin Hitam


CoS 8436CoS 91230

BarleyRice

Maize

Wheat


NarengaMiscanthus

N58

Oat

Sclerostachya

III

III









4 Conclusion


Competing Interests


Acknowledgments


References

















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


ble2Detailsof


42ES

T-SSRmarkersin

twentygeno

typesb

elon

ging

tocerealsa

ndSaccharum

related

genera

andSaccharum

speciesa

ndtheirc

ultiv

arsLanes1

to20

representn

umbero

fbands

prod

uced

inwheatm

aizebarleyric

epearlm

illetoatSorghum

NarengaScle

rosta

chyaEria

nthu

sMiscanthusB

andjermasin

Hita

mG

unjer

a51NG56N

58

CoS

92423CoS

88230UP9530C

oS91230andCoS

8436respectively

Snu

mberlane

12

34

56

78

910

1112

1314

1516

1718

1920

Prim

erpo

lymorph

ism(

)SY

284

56

12

30

25

53

23

23

31

32

395

SY29

63

59

312

35

126

77

87

76

76

68

100

SY30

10

40

07

05

66

33

43

23

23

23

80SY

310

00

00

00

23

21

12

22

02

20

050

SY32

10

01

02

02

43

13

00

10

00

00

55SY

330

02

23

53

76

46

55

52

30

23

385

SY34

00

00

00

00

00

03

25

45

55

99

85SY

350

00

10

40

52

10

01

11

22

12

165

SY36

00

00

00

00

30

05

22

20

20

02

35SY

379

75

66

55

912

1011

88

88

67

65

6100

SY38

00

03

02

07

81

00

22

12

00

30

50SY

450

00

00

20

51

01

21

10

00

00

035

SY46

01

44

10

04

40

10

00

00

00

00

35SY

470

13

32

00

10

00

00

00

00

00

025

SY48

00

00

53

08

120

53

32

13

22

00

60SY

530

00

11

33

129

21

12

21

32

13

185

SY58

00

01

10

32

10

00

00

00

00

00

25SY

590

00

00

00

40

33

00

10

00

00

020

SY61

03

75

40

34

56

34

15

35

55

55

90SY

620

00

10

20

45

32

20

00

00

00

035

SY63

00

00

00

01

14

16

11

10

10

21

55SY

650

00

00

00

00

11

10

12

32

22

150

SY66

00

00

00

01

00

03

12

00

32

21

40SY

671

25

63

30

25

63

20

02

00

00

060

SY69

15

20

10

03

12

11

11

12

10

32

80SY

701

00

10

10

52

00

10

02

03

30

045

SY72

10

20

00

01

45

08

58

13

64

68

70SY

730

02

01

20

22

01

31

41

03

00

360

SY74

21

10

11

31

32

14

15

10

30

02

80SY

751

02

33

00

06

39

76

76

04

60

065

SY76

00

00

02

01

21

40

00

10

00

00

30SY

772

56

68

43

96

93

04

43

43

02

085

SY78

22

56

46

45

76

410

910

32

87

59

100

SY79

00

00

10

03

52

05

17

72

10

06

55SY

801

10

40

10

45

62

54

55

35

45

585

SY81

00

01

26

06

68

73

34

33

33

32

80SY

820

010

31

43

91

16

54

35

34

43

290

SY83

12

06

23

42

72

13

23

13

32

33

95

SY84

00

01

23

00

20

03

31

03

33

33

60


Table2Con

tinued

Snu

mberlane

12

34

56

78

910

1112

1314

1516

1718

1920

Prim

erpo

lymorph

ism(

)SY

861

56

32

54

58

116

53

115

127

67

10100

SY89

96

54

17

37

43

104

45

73

47

35

100

SY90

50

32

52

34

34

04

36

44

23

64

90Av

erageo

ftransfe

rability

2722

2722

4722

4667

3611

5556

2611

8833

9889

7111

600

7333

5556

7556

550

5056

5889

5111

5278

600


2019181716151413121110987654321M

50bp

100 bp

200 bp300bp400bp

600bp1050bp1350bp


CoS 92423

51NG56

Bandjermasin Hitam


CoS 8436CoS 91230

BarleyRice

Maize

Wheat


NarengaMiscanthus

N58

Oat

Sclerostachya

III

III









4 Conclusion


Competing Interests


Acknowledgments


References

















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table2Con

tinued

Snu

mberlane

12

34

56

78

910

1112

1314

1516

1718

1920

Prim

erpo

lymorph

ism(

)SY

861

56

32

54

58

116

53

115

127

67

10100

SY89

96

54

17

37

43

104

45

73

47

35

100

SY90

50

32

52

34

34

04

36

44

23

64

90Av

erageo

ftransfe

rability

2722

2722

4722

4667

3611

5556

2611

8833

9889

7111

600

7333

5556

7556

550

5056

5889

5111

5278

600


2019181716151413121110987654321M

50bp

100 bp

200 bp300bp400bp

600bp1050bp1350bp


CoS 92423

51NG56

Bandjermasin Hitam


CoS 8436CoS 91230

BarleyRice

Maize

Wheat


NarengaMiscanthus

N58

Oat

Sclerostachya

III

III









4 Conclusion


Competing Interests


Acknowledgments


References

















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


2019181716151413121110987654321M

50bp

100 bp

200 bp300bp400bp

600bp1050bp1350bp


CoS 92423

51NG56

Bandjermasin Hitam


CoS 8436CoS 91230

BarleyRice

Maize

Wheat


NarengaMiscanthus

N58

Oat

Sclerostachya

III

III









4 Conclusion


Competing Interests


Acknowledgments


References

















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


4 Conclusion


Competing Interests


Acknowledgments


References

















































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



























































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Research Article Assessment of Functional EST-SSR Markers...

Documents

Transcript of Research Article Assessment of Functional EST-SSR Markers...